Ionic self-assembling peptides

ABSTRACT

Provided herein are ionic self-assembling peptides, pharmaceutical compositions comprising the peptides, and methods of using and making the same.

CLAIM OF PRIORITY

This application is a continuation of U.S. Nonprovisional applicationSer. No. 16/503,039, filed Jul. 3, 2019, which claims the benefit ofU.S. Provisional Application No. 62/693,877, filed Jul. 3, 2018. Theentire contents of the foregoing are incorporated herein by reference.

SEQUENCE LISTING

The instant application contains an XML Sequence Listing which has beensubmitted electronically and is hereby incorporated by reference in itsentirety. The Sequence Listing, created on Feb. 7, 2023 (EST), is named3DMJP-NA001US04-SL.xml and is 158,883 bytes in size.

FIELD OF THE INVENTION

The disclosure generally relates to self-assembling peptides,pharmaceutical compositions comprising the self-assembling peptides, andmethods of using the same.

BACKGROUND

Self-assembling peptides have been developed for various purposes,including scaffolding for tissue engineering and regenerative medicine,drug delivery, three-dimensional tissue culture, and hemostasis.Examples of such self-assembling peptides include β-sheet peptides (1)with alternating positively and negatively charges and hydrophobicresidues (e.g., RADA16 (SEQ ID NO:91), IEIK13 (SEQ ID NO:92), and KLDL12(SEQ ID NO:93)), (2) with alternating sequences of non-ionic, polarresidues and hydrophobic residues, and (3) with repeating non-ionic,polar residues. However, such self-assembling peptides have practicallimitations. For example, pharmaceutical compositions including theseself-assembling peptides with alternating positively and negativelycharges and hydrophobic residues (e.g., RADA16 (SEQ ID NO:91)) must beformulated at acidic pH in order to solubilize the peptides, which cancause cell and/or tissue damage upon administration to a subject. Thus,there remains a need for improved self-assembling peptides that can beused for therapeutic applications.

SUMMARY OF THE INVENTION

The present disclosure is based, at least in part, on the development ofionic self-assembling peptides having specific combinations of ionicpolar amino acids, hydrophobic amino acids, and non-ionic polar aminoacids. Previously described self-assembling peptides typically includeequivalent amounts of amino acid residues having net-negative charge andnet-positive charge at neutral pH. These peptides are formulated atacidic or basic pH to charge the peptides such that the peptidesremained in solution, and such that the compositions comprising thepeptides were flowable and injectable. If formulated at neutral pH,phase separation and precipitation of the peptides was observed. Forexample, RADA16 (SEQ ID NO:91) has four positive charges and fournegative charges at neutral pH. As shown in FIG. 20A, when formulated atpH 7.5 RADA 16 (SEQ ID NO:91) precipitates.

In contrast, the inclusion of ionic polar amino acids in theself-assembling peptides provided herein results in peptides havingnon-zero net charge at neutral pH, thereby allowing for the peptides tobe formulated at neutral pH. Advantageously, the ionic self-assemblingpeptides provided herein can be formulated at neutral pH and remainsoluble and stable in solution. Without wishing to be bound by anyparticular theory, pharmaceutical compositions comprising the ionicself-assembling peptides described herein may be formulated at neutralpH and administered to a subject without inducing tissue damage that maybe associated with pharmaceutical compositions having acidic or basicpH. Moreover, the ionic self-assembling peptides can be used to makeimproved hydrogels having advantageous physical characteristics underphysiological conditions. For example, aqueous solutions of theself-assembling peptides may be produced which remain stable, and areflowable and injectable under physiological conditions. Upon gelation,the self-assembling peptides may form hydrogels having shear-thinning,thixotropic, and rheological properties that are useful in clinical,industrial, and/or research applications.

In some aspects, the disclosure provides self-assembling peptides andpharmaceutical compositions comprising the self-self assemblingpeptides, wherein the self-assembling peptide comprises or consist of anamino acid sequence as set forth in: [(X)i(Y)j(Z)k(Y)l]m(X)n (FormulaI), [(Y)i(X)j(Y)k(Z)l]m(Y)n (Formula II), [(Z)i(Y)j(X)k(Y)l]m(Z)n(Formula III), or [(Y)i(Z)j(Y)k(X)l]m(Y)n (Formula IV), wherein each (X)is independently an ionic, polar amino acid, wherein each (Y) isindependently a hydrophobic amino acid, wherein each (Z) isindependently a non-ionic, polar amino acid, wherein each i, j, k, and lis independently an integer ≥1, wherein m is an integer ≥2, and whereinn=0 or an integer ≥1. In some embodiments, at least one of i, j, k, andl is independently an integer of 1. In some embodiments, at least one ofi, j, k, and l is independently an integer of 2. In some embodiments,each of i, j, k, and l is 1. In some embodiments, m is independently aninteger of 2, 3, or 4.

In some embodiments, the self-assembling peptide comprises or consistsof an amino acid sequence as set forth in Formula I. In someembodiments, the self-assembling peptide comprises or consists of anamino acid sequence as set forth in Formula II. In some embodiments, theself-assembling peptide comprises or consists of an amino acids sequenceas set forth in Formula III. In some embodiments, the self-assemblingpeptide comprises or consists of an amino acid sequence as set forth inFormula IV.

In some embodiments, each (X) is a basic amino acid (e.g., arginine,lysine, histidine, or ornithine). In some embodiments, each (X) is anacidic amino acid (e.g., aspartic acid or glutamic acid).

In some embodiments, each (Y) is either alanine, valine, leucine,isoleucine, methionine, phenylalanine, tryptophan, or glycine.

In some embodiments, each (Z) is either serine, threonine, tyrosine,cysteine, glutamine, asparagine, or methionine.

In some embodiments, the self-assembling peptide comprises or consistsof an amino acid sequence as set forth in SEQ ID NOs:1-20 and 94-96.

In some embodiments, the self-assembling peptide comprises an N-terminalfunctional group, a C-terminal functional group, or both. In someembodiments, the N-terminal functional group is either an acetyl, aformyl, pyroglutamyl (pGlu), biotin, polyethylene glycol (PEG), urea,alkylamine, a carbamate, a sulfonamide, dansyl, 2,4-dintrophenyl,fluorescein, 7-methoxycoumarin acetic acid,9-fluorenylmethyloxycarbonyl, palmitic acid, succinyl, chloroacetyl,maleimide, benzyloxycarbonyl, bromoacetyl, nitrilotriacetyl,tertbutoxycarbonyl, 4-hydroxyphenylpropionic acid, allyloxycarbonyl,butyric acid, a fatty acid, or trityl. In some embodiments, theC-terminal functional group is either an amido, an N-alkyl amide, analdehyde, an ester, an alcohol, para-nitroanilide (pNA),7-amino-4-methylcoumarin (Amc), a hydrazide, hydroxamic acid,chloromethylketone, p-nitroaniline, para-nitrophenol, hydroxysucinimideester, fluoromethylketone, cysteamide, 9-fluorenemethyl (Fm) ester,allyl ester, 2,4-dimethoxybenzyl ester, 2-phenylisopropyl ester,p-nitrobenzyl ester, and 2-chlorotrityl ester. In some embodiments, theself-assembling peptide comprises or consists of an amino acid sequenceas set forth in SEQ ID NOs:21-40 and 97-99.

In some embodiments, the self-assembling peptide comprises at least one(e.g., one, two, three, four, or more) biologically active peptidemotif. In some embodiments, the at least one biologically active peptidemotif is present at the N-terminal end of the self-assembling peptide.In some embodiments, the at least one biologically-active peptide motifis present at the C-terminal end of the self-assembling peptide. In someembodiments, the at least one biologically-active peptide motif isderived from laminin-1, collagen IV, fibronectin, elastin, bone marrowhoming peptide 1, bone marrow homing peptide 2, or myelopeptide. In someembodiments, the at least one biologically-active peptide motifcomprises or consists of an amino acid sequence as set forth in any oneof SEQ ID NOs:41-70. In some embodiments, the self-assembling peptidecomprises or consists of an amino acid sequence as set forth in SEQ IDNOs:71-90.

In some embodiments, the pharmaceutical composition comprises a tonicityagent. In some embodiments, the tonicity agent comprises one or moresalts selected from the group consisting of NaCl, KCl, MgCl₂, CaCl₂,NH₄Cl, Na₂HPO₄, KH₂PO₄, and CaSO₄. In some embodiments, the tonicityagent further comprises one or more sugars selected from the groupconsisting of dextrose, mannitol, glycerin, sucrose, and trehalose. Insome embodiments (e.g., when the tonicity agent is one or more salts),the tonicity agent is present at a concentration of about 0.01 M toabout 0.3 M. In some embodiments (e.g., when the tonicity agent is oneor more salts), the tonicity agent is present at a concentration ofabout 0.15 M. In some embodiments (e.g., when the tonicity agent is oneor more sugars), the tonicity agent is present at a concentration ofabout 0.1 to 10% (w/v). In some embodiments (e.g., when the tonicityagent is one or more sugars), the tonicity agent is present at aconcentration of about 10% (w/v). In some embodiments, the tonicityagent increases rheological properties of the composition or hydrogelpeptide comprising the self-assembling peptides described herein.

In some embodiments, the pharmaceutical composition has a pH of fromabout 6 to about 8. In some embodiments, the pharmaceutical compositionhas a pH of from about 7 to about 7.5. In some embodiments, the netcharge of the self-assembling peptide in the pharmaceutical compositionis greater than or equal to +1 or less than or equal to −1. In someembodiments, the net charge of the self-assembling peptide in thepharmaceutical composition is from about +1 to about +6 (e.g., +1, +2,+3, +4, +5, or +6). In some embodiments, the net charge of theself-assembling peptide in the pharmaceutical composition is from about−1 to about −6 (e.g., −1, −2, −3, −4, −5, or −6).

In some embodiments, the concentration of the self-assembling peptide isthe pharmaceutical composition is from about 0.01% (w/v) to about 10%(w/v). In some embodiments, the concentration of the self-assemblingpeptide is the pharmaceutical composition is from about 0.1% (w/v) toabout 5% (w/v). In some embodiments, the concentration of theself-assembling peptide is the pharmaceutical composition is from about0.5% (w/v) to about 1.5% (w/v). In some embodiments, the concentrationof the self-assembling peptide is the pharmaceutical composition isabout 1% (w/v).

In some embodiments, the pharmaceutical composition comprises anisolated cell (e.g., a stem cell). In some embodiments, the isolatedcell is a mammalian cell. In some embodiments, the mammalian cell is animmune cell, a stem cell, chondrocyte progenitor cells, pancreaticprogenitor cells, myoblasts, fibroblasts, keratinocytes, neuronal cells,glial cells, astrocytes, pre-adipocytes, adipocytes, vascularendothelial cells, endothelial progenitor cells, mesenchymal cells,neural stem cells, immune cells, (e.g., B-cells and T-cells), smoothmuscle progenitor cells, cardiac myocytes, fetal dermal fibroblasts,epidermal keratinocytes, myoblasts, and capillary endothelial cells.

In some embodiments, the pharmaceutical composition comprises at leastone (e.g., one, two, three, four, five, or more) bioactive agent. Insome embodiments, the bioactive agent is a hormone, a growth factor,insulin, an enzyme, an siRNA, an shRNA, an antisense-RNA, ananti-sense-DNA, an mRNA, an antibiotic, an antibody, or ananti-inflammatory agent.

In some embodiments, the pharmaceutical composition is an aqueoussolution. In some embodiments, the pharmaceutical composition is ahydrogel.

In some embodiments, the pharmaceutical composition is a hydrogel havinga storage modulus of at least about 10 Pascal (Pa) (e.g., about 25 Pa,about 50 Pa, about 100 Pa, about 150 Pa, about 250 Pa, about 500 Pa,about 750 Pa, about 1000 Pa, or more).

In another aspect, the disclosure provides articles of manufacturecomprising the self-assembling peptides or the pharmaceuticalcomposition described herein. In some embodiments, the article is asyringe, a vial, an auto-injector, tubing, or a catheter.

In another aspect, the disclosure provides methods of treating a subjectin need thereof, comprising administering an effective amount of aself-assembling peptide or of a pharmaceutical composition describedherein to the subject.

In yet another aspect, the disclosure provides methods of promotingtissue repair or regeneration in a subject in need thereof, comprisingcontacting a tissue of the subject with a self-assembling peptide or apharmaceutical composition described herein, thereby promoting tissuerepair or regeneration of the tissue. In some embodiments, the tissue isskin, bone, cartilage, neural tissue, ligament, tendon, vascular tissue,or muscle. In some embodiments, the tissue is optic tissue. In someembodiments, the tissue is cardiac tissue. In some embodiments, thesubject has a congenital disease or disorder resulting in a need for thetissue repair or regeneration. In some embodiments, the subject hassuffered an injury (e.g., surgery, trauma, stroke, tumor, or a diseaseor disorder) resulting in a need for the tissue repair or regeneration.

In another aspect, the disclosure provides methods of promoting woundhealing in a subject in need thereof, comprising contacting a wound ofthe subject with (or, alternatively, administering to a subject's wound)an effective amount of a self-assembling peptide or a pharmaceuticalcomposition described herein, thereby promoting wound healing and/orantimicrobial activity. In some embodiments, the wound comprises orconsists of an abrasion, a burn, a chap, a detrition, a cut, an ulcer, alaceration, an incision, or a scratch.

In another aspect, the disclosure provides methods of stopping orpreventing (or, alternatively, reducing) bleeding at a site within asubject, comprising contacting the site with (or, alternatively,administering to a subject's wound) a self-assembling peptide or apharmaceutical composition described herein, thereby creating a physicalbarrier thereby stopping or preventing (or reducing) bleeding at thesite within the subject.

In another aspect, the disclosure provides methods of excising a lesionfrom a site in the gastrointestinal tract of a subject, comprisingcontacting submucosa below the lesion with (or, alternatively,administering to the submucosa below the lesion) a self-assemblingpeptide or a pharmaceutical composition described herein, in an amountsufficient to lift the lesion; and excising the lesion from the site inthe gastrointestinal tract of the subject. In some embodiments, thelesion comprises a polyp, an ulcer, or a tumor. In some embodiments, thelesion is present in a region of the gastrointestinal tract of thesubject selected from a mouth, a throat, an esophagus, a stomach, asmall intestine, a large intestine, a colon and a rectum.

In yet another aspect, the disclosure provides methods of culturing acell that include contacting the cell with a pharmaceutical compositiondescribed herein.

In another aspect, provided herein is a method of treating a pulmonarybulla in a subject, comprising:

introducing a delivery device to a target area of the pulmonary bulla ofthe subject; positioning an end of the delivery device in the targetarea in which a treatment of the pulmonary bulla is desired;

administering, through the delivery device, a self-assembling peptide ora pharmaceutical composition described herein in an effective amount andin an effective concentration to the target area to form a barrier underphysiological conditions of the target area to treat the pulmonarybulla;

removing the delivery device from the target area; and

collapsing the pulmonary bulla prior or subsequent to administering thesolution.

In another aspect, provided herein is a method for mitigating adhesionto a biological tissue, the method comprising administering to thebiological tissue an effective amount of a self-assembling peptide or apharmaceutical composition described herein, to thereby mitigateadhesion to the biological tissue.

In another aspect, provided herein is a method of filling a bone void ina subject, comprising

introducing a delivery device to a bone of a subject;

positioning an end of the delivery device proximate a void in the bonewhere promotion of bone growth is desired;

administering a self-assembling peptide or a pharmaceutical compositiondescribed herein in a concentration sufficient to form a hydrogelscaffold under physiological conditions through the delivery device; and

removing the delivery device.

In another aspect, provided herein is a method of treating dry eye in asubject, comprising administering to an eye of the subject an effectiveamount of a self-assembling peptide or a pharmaceutical compositiondescribed herein.

The present invention further provides self-assembling peptidescomprising or consisting of an amino acid sequence as set forth in:[(X)i(Y)j(Z)k(Y)l]m(X)n (Formula I), [(Y)i(X)j(Y)k(Z)l]m(Y)n (FormulaII), [(Z)i(Y)j(X)k(Y)l]m(Z)n (Formula III), or [(Y)i(Z)j(Y)k(X)l]m(Y)n(Formula IV), for use in any of the methods described herein.

Other features and advantages of the invention will be apparent from thefollowing detailed description and figures, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows net charge measurements of KLNL12 (SEQ ID NO:21) at variouspH.

FIG. 2 shows net charge measurements of NLEL12 (SEQ ID NO:33) at variouspH.

FIG. 3 shows net charge measurements of RADA16 (SEQ ID NO:91) at variouspH.

FIG. 4 shows a schematic representation of the molecular structure andthe electronic charge of RANA16 (SEQ ID NO:39) at pH 7.5.

FIG. 5 shows a schematic representation of the molecular structure andthe electronic charge of RADA16 (SEQ ID NO:91) at pH 7.5.

FIGS. 6-8 show shear-thinning properties of exemplary aqueouspharmaceutical compositions including the self-assembling peptidesKLNL12 (SEQ ID NO:21), IQIK13 (SEQ ID NO:28), or NLEL12 (SEQ ID NO:33).

FIG. 6 shows shear-thinning properties of an aqueous pharmaceuticalcompositions including 1% (w/v) KLNL12 (SEQ ID NO:21) at pH 7.5, asreflected by changing viscosity in response to various shear rates.

FIG. 7 shows shear-thinning properties of an aqueous pharmaceuticalcompositions including 1% (w/v) IQIK13 (SEQ ID NO:28) at pH 7.5, asreflected by changing viscosity in response to various shear rates.

FIG. 8 shows shear-thinning properties of an aqueous pharmaceuticalcompositions including 1% (w/v) NLEL12 (SEQ ID NO:33) at pH 7.5, asreflected by changing viscosity in response to various shear rates.

FIGS. 9-10 show shear-thinning properties of exemplary aqueouspharmaceutical compositions including the self-assembling peptidesKLNL12 (SEQ ID NO:21) or NLEL12 (SEQ ID NO:33).

FIG. 9 shows shear-thinning properties of an aqueous pharmaceuticalcompositions including 1% (w/v) KLNL12 (SEQ ID NO:21) containing 0.9%NaCl at pH 7.5, as reflected by changing viscosity in response tovarious shear rates.

FIG. 10 shows shear-thinning properties of an aqueous pharmaceuticalcompositions including 1% NLEL12 (SEQ ID NO:33) containing 0.9% NaCl atpH 7.5, as reflected by changing viscosity in response to various shearrates.

FIGS. 11-12 show thixotropic properties of exemplary aqueouspharmaceutical compositions including the self-assembling peptidesKLNL12 (SEQ ID NO:21) or NLEL12 (SEQ ID NO:33).

FIG. 11 shows thixotropic properties of an aqueous pharmaceuticalcompositions including 1% (w/v) KLNL12 (SEQ ID NO:21) containing 0.9%NaCl at pH 7.5, as reflected by changes in mechanical strength aftershear stress is removed.

FIG. 12 shows thixotropic properties of an aqueous pharmaceuticalcompositions including 1% NLEL12 (SEQ ID NO:33) containing 0.9% NaCl atpH 7.5, as reflected by changes in mechanical strength after shearstress is removed.

FIGS. 13-15 show changes in rheological properties of exemplary aqueouspharmaceutical compositions including the self-assembling peptidesKLNL12 (SEQ ID NO:21), KIQI13 (SEQ ID NO:29), or NLEL12 (SEQ ID NO:33),in response to exposure to DMEM.

FIG. 13 shows changes in rheological properties of an aqueouspharmaceutical compositions including 1% (w/v) KLNL12 (SEQ ID NO:21) atpH 7.5, as reflected by increased mechanical strength after exposure toDMEM.

FIG. 14 shows changes in rheological properties of an aqueouspharmaceutical compositions including 1% (w/v) KIQI13 (SEQ ID NO:29) atpH 7.5, as reflected by increased mechanical strength after exposure toDMEM.

FIG. 15 shows changes in rheological properties an aqueouspharmaceutical compositions including 1% (w/v) NLEL12 (SEQ ID NO:33) atpH 7.5, as reflected by increased mechanical strength after exposure toDMEM.

FIGS. 16-18 show changes in rheological properties of exemplary aqueouspharmaceutical compositions including the self-assembling peptidesKLNL12 (SEQ ID NO:21), KIQI13 (SEQ ID NO:29), or NLEL12 (SEQ ID NO:33),in response to exposure to DMEM.

FIG. 16 shows changes in rheological properties of an aqueouspharmaceutical compositions including 1% (w/v) KLNL12 (SEQ ID NO:21)containing 0.9% NaCl at pH 7.5, as reflected by increased mechanicalstrength after exposure to DMEM.

FIG. 17 shows changes in rheological properties of an aqueouspharmaceutical compositions including 1% (w/v) KIQI13 (SEQ ID NO:29)containing 0.9% NaCl at pH 7.5, as reflected by increased mechanicalstrength after exposure to DMEM.

FIG. 18 shows changes in rheological properties of an aqueouspharmaceutical compositions including 1% NLEL12 (SEQ ID NO:33)containing 0.9% NaCl at pH 7.5, as reflected by increased mechanicalstrength after exposure to DMEM.

FIG. 19 is a schematic representation of macromolecular structures thatcan be formed by the self-assembling peptide of the invention.

FIGS. 20A-20C below are photographs showing the appearance of aqueouspharmaceutical compositions including the specified self-assemblingpeptide at pH 7.5. FIG. 20A is a photograph showing the appearance of anaqueous pharmaceutical composition including RADA16 (SEQ ID NO:91), pH7.5.

FIG. 20B is a photograph showing the appearance of an aqueouspharmaceutical composition including KLNL12 (SEQ ID NO:21), pH 7.5.

FIG. 20C is a photograph showing the appearance of an aqueouspharmaceutical composition including NLEL12 (SEQ ID NO:33), pH 7.5.

FIGS. 21A and 21B show the appearance of pig stomach submucosa followinginjections of the specified pharmaceutical compositions after 0-5minutes (FIG. 21A) and after 15-20 minutes (FIG. 21B). Injection sitesare indicated with dotted black ovals: (1) 2 mL of 0.1% (w/v) KLNL12(SEQ ID NO:21), 0.9% (w/v) NaCl pH 7.5; (2) 2 mL of 0.1% (w/v) NLKL12(SEQ ID NO:23), 0.9% (w/v) NaCl pH 7.5; (3) 2 mL of 0.1% (w/v) KIQI13(SEQ ID NO:29), 0.9% (w/v) NaCl pH 7.5; (4) 2 mL of 0.2% (w/v) RADA16(SEQ ID NO:91) pH 2.5; (5) 2 mL of MucoUp®; (6) 2 mL of saline pH 7.5.

FIGS. 22A-22C show the appearance of dissected pig stomach submucosafollowing injections of either 2 mL saline at pH 7.5 (FIG. 22A), or 2 mLof an aqueous pharmaceutical composition including 0.2% (w/v) RADA16(SEQ ID NO:91) at pH 2.5 (FIG. 22B), or 2 ml of an aqueouspharmaceutical composition including 0.2% (w/v) RADA16 (SEQ ID NO:91),0.9% NaCl (w/v) at pH 2.5 (FIG. 22C). Black ovals indicate areas thatwere white and had a cloudy undefined appearance.

FIGS. 23A, 23B, 23C, and 23D show the appearance of pig stomachsubmucosa following injections of 2 mL of a pharmaceutical compositionincluding 0.1% KLNL12 (SEQ ID NO:21), 0.9% NaCl, at pH 7.5 (FIG. 23A); 2mL of a pharmaceutical composition including 0.2% NLEL12 (SEQ ID NO:33),0.9% NaCl, at pH 7.5 (FIG. 23B); 2 mL of a pharmaceutical compositionincluding 0.2% QLEL12 (SEQ ID NO:35), 0.9% NaCl, at pH 7.5 (FIG. 23C);or 2 mL of a pharmaceutical composition including 0.2% LELQ12 (SEQ IDNO:36), 0.9% NaCl, at pH 7.5.

FIG. 24A shows changes in rheological properties of an aqueouspharmaceutical compositions including 0.15% (w/v) QLEL12 (SEQ ID NO:35)with 0.9% NaCl (w/v) at pH 7.5, as reflected by increased mechanicalstrength after exposure to DMEM.

FIG. 24B shows rheological properties of an aqueous pharmaceuticalcompositions including QLEL12 (SEQ ID NO:35) with 0.9% NaCl (w/v) at pH7.5 at various concentrations between 0.1% (w/v) and 0.3% (w/v); linearregression was performed.

FIGS. 25A-25D shows elevation heights and lifting capability of thesubmucosal layer in the canine stomach and colon after injecting 0.5 mLof QLEL12 (SEQ ID NO:35) with 0.9% NaCl (w/v) at pH 7.5 at variousconcentrations between 0.1% (w/v) and 0.3% (w/v). These and otheradvantages of the present technology will be apparent when reference ismade to the following description.

DETAILED DESCRIPTION Definitions

Unless otherwise defined herein, scientific and technical terms used inthis disclosure shall have the meanings that are commonly understood bythose of ordinary skill in the art. Generally, nomenclature used inconnection with, and techniques of, chemistry, cell and tissue culture,molecular biology, cell and cancer biology, neurobiology,neurochemistry, virology, immunology, microbiology, pharmacology,genetics and protein and nucleic acid chemistry, described herein, arethose well-known and commonly used in the art.

The methods and techniques of the present disclosure are generallyperformed, unless otherwise indicated, according to conventional methodswell known in the art and as described in various general and morespecific references that are cited and discussed throughout thisspecification.

Chemistry terms used herein are used according to conventional usage inthe art, as exemplified by “The McGraw-Hill Dictionary of ChemicalTerms,” Parker S., Ed., McGraw-Hill, San Francisco, Calif. (1985).

All of the publications, patents and published patent disclosuresreferred to in this disclosure are specifically incorporated byreference herein. In case of conflict, the present specification,including its specific definitions, will control.

The term “self-assembling”, as used herein, refers to the ability ofcertain peptides to spontaneously self-associate into higher orderstructures (e.g., β-sheets). For example, pharmaceutical compositionscomprising self-assembling peptides in the form of a solution transitionto a gel state when the self-assembling peptides self-associate. In someembodiments, the interaction between and among individualself-assembling peptides are reversible, such that the composition mayreversibly transition between a gel state and a solution state. Theinteractions between and among individual self-assembling peptides maybe non-covalent interactions including hydrogen bonds, ionicinteractions, electrostatic interactions (e.g., via van der Waalsforces), and hydrophobic interactions. In various embodiments, theself-assembled peptide nanostructure comprises peptides in β-sheets. Thenanostructure can be a nanofiber, or a network of nanofibers. Forillustrative purposes, FIG. 19 shows the assembly of self-assemblingpeptides into (3 sheets 1901. These (3 sheets can self-assemble into ananofiber 1902. A plurality of nanofibers 1902 can self-assemble into amembrane network 1903. In some embodiments, the self-assembly of thepeptides described herein into higher order structures is responsive toone or more environmental triggers (e.g., change in one or more of pH,temperature, ionic strength, osmolarity, osmolality, applied pressure,applied shear stress, etc.).

As used herein, the term “administering” is intended to include, but isnot limited to, applying, introducing or injecting a self-assemblingpeptide and/or a pharmaceutical composition comprising a self-assemblingpeptide described herein.

As used herein, the term “amino acid residue” or “amino acid” includesnatural and synthetic amino acid residues including D- and L-aminoacids; alpha-, beta- and gamma-amino acids; chemically-modified aminoacids; naturally-occurring non-proteogenic amino acids; rare aminoacids; and chemically synthesized compounds that have properties knownin the art to be characteristic of an amino acid.

As used herein, the phrases “therapeutically effective amount”,“effective amount” or “effective dose” refer to an amount ofpharmaceutical composition that when delivered to a subject provides atherapeutic or aesthetic benefit in the treatment, prevention, ormanagement of a tissue, disease and/or disorder. Determination of atherapeutically effective amount is well within the capability of thoseskilled in the art. Generally, a therapeutically effective amount canvary with the subject's history, age, condition, sex, as well as theseverity and type of the medical condition in the subject, andadministration of other pharmaceutically active agents.

As used herein, the term “hydrogel” refers to a composition comprising athree dimensional network of self-assembling peptides. The term hydrogelmay be used to refer to a network of self-assembling peptides in a drystate (xerogel) or in a wet state. In a wet state, the hydrogel mayinclude a high water content (e.g., between about 90% to about 99.9%water). Hydrogels have many desirable properties for biomedicalapplications. For example, hydrogels can be manufactured such that theyare nontoxic and compatible with tissue. In addition, hydrogels areusually highly permeable to water, ions, and small molecules.

As used herein the term “isolated” in reference to cells refers to acell that has been mechanically separated from the environment fromwhich it is found in nature.

As used herein, the term “tonicity agent” refers to an agent which maybe used to regulate the osmotic pressure of the pharmaceuticalcompositions described herein. Exemplary tonicity agents, include, butare not limited to sodium chloride, calcium chloride, potassiumchloride, potassium phosphate, and sugars (e.g., dextrose and sucrose).In some embodiments, the tonicity agent is sodium chloride. The tonicityagent can increase rheological properties of a solution or composition(e.g., hydrogel) comprising the self-assembling peptides disclosedherein.

A “pharmaceutical composition,” as used herein, refers to a compositioncomprising a self-assembling peptide and other components, such as aphysiologically suitable carrier and/or excipient.

As used herein, the terms “proteins” and “peptides” are usedinterchangeably to refer to a polymer of amino acid residues connectedto the other by peptide bonds between the alpha-amino and carboxy groupsof adjacent residues. The terms “protein”, and “peptide include polymershaving modified amino acid residues (e.g., phosphorylated, amidated, orglycated amino acid residues) and amino acid analogs.

As used herein, the term “subject” refers to either a human or non-humananimal. The term includes, but is not limited to, mammals (e.g., humans,other primates, pigs, rodents (e.g., mice and rats or hamsters),rabbits, guinea pigs, cows, horses, cats, dogs, sheep, and goats). Insome embodiments, the subject is a human. In some embodiments, thesubject is an adult human subject. In some embodiments, the subject is apediatric human subject.

The term “wound” used herein refers to a trauma in a tissue of a subject(e.g., a human subject) such as an abrasion, a burn, a chap, adetrition, a cut, an ulcer, a laceration, an incision, or a scratch.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting. Citation or identification of any document in thisapplication is not an admission that such document is available as priorart to the present invention.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

Pharmaceutical Compositions and Self-Assembling Peptides

In one aspect, the disclosure provides self-assembling peptides andpharmaceutical compositions comprising at least one self-assemblingpeptide described herein. The self-assembling peptides spontaneouslyassemble into higher order structures via intermolecular andintramolecular electrostatic interactions when present in an aqueoussolution. These higher order structures include β-sheets, nanofiberstructures, and three-dimensional network or mesh structures. When thesehigher order structures form aqueous solutions may become hydrogelshaving a variety of desirable properties. In addition to theself-assembling peptides, the pharmaceutical compositions may alsoinclude other additives such as tonicity agents, buffers,pharmaceutically-acceptable excipients, biomolecules, therapeuticagents, and cells.

In some embodiment, the self-assembling peptides are biodegradable. Asused herein, “biodegradable” refers to materials that degrade or breakdown upon interaction with a physiological environment into componentsthat can be metabolized or excreted by a subject over a period of timeranging from minutes to years (e.g., one day, two days, three days, fourdays, five days, six days, seven days, two weeks, three weeks, fourweeks, two months, three months, six months, one year, two years, ormore). In some embodiments, the self-assembling peptides may degrade viacleavage of the peptide chain, for example, via hydrolysis or enzymaticcleavage. Although the self-assembling peptides (and higher orderstructures (e.g., scaffolds) formed by the self-assembling peptides) maybe biodegradable, these higher order structures preferably maintaintheir structural integrity for a period of time required for theirintended use.

The self-assembling peptides provided herein comprise or consist of anamino acid sequence as set forth in

[(X)i(Y)j(Z)k(Y)l]m(X)n  (Formula I),

[(Y)i(X)j(Y)k(Z)l]m(Y)n  (Formula II),

[(Z)i(Y)j(X)k(Y)l]m(Z)n  (Formula III), or

[(Y)i(Z)j(Y)k(X)l]m(Y)n  (Formula IV),

wherein each (X) is independently an ionic, polar amino acid, whereineach (Y) is independently a hydrophobic amino acid, wherein each (Z) isindependently a non-ionic, polar amino acid, wherein each i, j, k, and lis independently an integer ≥1, wherein m is an integer ≥2, and whereinn=0 or an integer ≥1. In some embodiments, each i, j, k, and l isindependently an integer of 1. In some embodiments, each i, j, k, and lis independently an integer of 2. In some embodiments, each i, j, k, andl is independently an integer of 3. In some embodiments, n is 0. In someembodiments, n is an integer ≥1 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, or more). In some embodiments, m is an integer of 1. In someembodiments, m is an integer ≥2 (e.g., 2, 3, 4, 6, 7, 8, 9, 10, 11, 12,or more). In some embodiments, m is an integer of 2, 3, or 4. In someembodiments, each (X) is the same amino acid, each (Y) is the same aminoacid, and/or each (Z) is the same type of amino acid residue.

In some embodiments, the self-assembling peptide comprises or consistsof an amino acid sequence as set forth in [(X)i(Y)j(Z)k(Y)l]m(X)n(Formula I), wherein each (X) is independently an ionic, polar aminoacid, wherein each (Y) is independently a hydrophobic amino acid,wherein each (Z) is independently a non-ionic, polar amino acid, whereineach i, j, k, and l is independently an integer ≥1, wherein m is aninteger ≥2, and wherein n=0 or an integer ≥1. In some embodiments, eachi, j, k, and l is independently an integer of 1. In some embodiments,each i, j, k, and l is independently an integer of 2. In someembodiments, each i, j, k, and l is independently an integer of 3. Insome embodiments, n is 0. In some embodiments, n is an integer ≥1 (e.g.,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more). In some embodiments, mis an integer of 1. In some embodiments, m is an integer ≥2 (e.g., 2, 3,4, 6, 7, 8, 9, 10, 11, 12, or more). In some embodiments, m is aninteger of 2, 3, or 4.

In some embodiments, the self-assembling peptide comprises or consistsof an amino acid sequence as set forth in [(Y)i(X)j(Y)k(Z)l]m(Y)n(Formula II), wherein each (X) is independently an ionic, polar aminoacid, wherein each (Y) is independently a hydrophobic amino acid,wherein each (Z) is independently a non-ionic, polar amino acid, whereineach i, j, k, and l is independently an integer ≥1, wherein m is aninteger ≥2, and wherein n=0 or an integer ≥1. In some embodiments, eachi, j, k, and l is independently an integer of 1. In some embodiments,each i, j, k, and l is independently an integer of 2. In someembodiments, each i, j, k, and l is independently an integer of 3. Insome embodiments, n is 0. In some embodiments, n is an integer ≥1 (e.g.,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more). In some embodiments, mis an integer of 1. In some embodiments, m is an integer ≥2 (e.g., 2, 3,4, 6, 7, 8, 9, 10, 11, 12, or more). In some embodiments, m is aninteger of 2, 3, or 4.

In some embodiments, the self-assembling peptide comprises or consistsof an amino acid sequence as set forth in [(Z)i(Y)j(X)k(Y)l]m(Z)n(Formula III), wherein each (X) is independently an ionic, polar aminoacid, wherein each (Y) is independently a hydrophobic amino acid,wherein each (Z) is independently a non-ionic, polar amino acid, whereineach i, j, k, and l is independently an integer ≥1, wherein m is aninteger ≥2, and wherein n=0 or an integer ≥1. In some embodiments, eachi, j, k, and l is independently an integer of 1. In some embodiments,each i, j, k, and l is independently an integer of 2. In someembodiments, each i, j, k, and l is independently an integer of 3. Insome embodiments, n is 0. In some embodiments, n is an integer ≥1 (e.g.,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more). In some embodiments, mis an integer of 1. In some embodiments, m is an integer ≥2 (e.g., 2, 3,4, 6, 7, 8, 9, 10, 11, 12, or more). In some embodiments, m is aninteger of 2, 3, or 4.

In some embodiments, the self-assembling peptide comprises or consistsof an amino acid sequence as set forth in [(Y)i(Z)j(Y)k(X)l]m(Y)n(Formula IV), wherein each (X) is independently an ionic, polar aminoacid, wherein each (Y) is independently a hydrophobic amino acid,wherein each (Z) is independently a non-ionic, polar amino acid, whereineach i, j, k, and l is independently an integer ≥1, wherein m is aninteger ≥2, and wherein n=0 or an integer ≥1. In some embodiments, eachi, j, k, and l is independently an integer of 1. In some embodiments,each i, j, k, and l is independently an integer of 2. In someembodiments, each i, j, k, and l is independently an integer of 3. Insome embodiments, n is 0. In some embodiments, n is an integer ≥1 (e.g.,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more). In some embodiments, mis an integer of 1. In some embodiments, m is an integer ≥2 (e.g., 2, 3,4, 6, 7, 8, 9, 10, 11, 12, or more). In some embodiments, m is aninteger of 2, 3, or 4.

In some embodiments, the self-assembling peptide comprises or consistsof an amino acid sequence as set forth in [(X)i(Y)j(Z)k(Y)l]m (FormulaV), wherein each (X) is independently an ionic, polar amino acid,wherein each (Y) is independently a hydrophobic amino acid, wherein each(Z) is independently a non-ionic, polar amino acid, wherein each i, j,k, and l is independently an integer ≥1, wherein m is an integer ≥2, andwherein n=0 or an integer ≥1. In some embodiments, each i, j, k, and lis independently an integer of 1. In some embodiments, each i, j, k, andl is independently an integer of 2. In some embodiments, each i, j, k,and l is independently an integer of 3. In some embodiments, n is 0. Insome embodiments, n is an integer ≥1 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, or more). In some embodiments, m is an integer of 1. In someembodiments, m is an integer ≥2 (e.g., 2, 3, 4, 6, 7, 8, 9, 10, 11, 12,or more). In some embodiments, m is an integer of 2, 3, or 4.

In some embodiments, the self-assembling peptide comprises or consistsof an amino acid sequence as set forth in [(Y)i(X)j(Y)k(Z)l]m (FormulaVI), wherein each (X) is independently an ionic, polar amino acid,wherein each (Y) is independently a hydrophobic amino acid, wherein each(Z) is independently a non-ionic, polar amino acid, wherein each i, j,k, and l is independently an integer ≥1, wherein m is an integer ≥2, andwherein n=0 or an integer ≥1. In some embodiments, each i, j, k, and lis independently an integer of 1. In some embodiments, each i, j, k, andl is independently an integer of 2. In some embodiments, each i, j, k,and l is independently an integer of 3. In some embodiments, n is 0. Insome embodiments, n is an integer ≥1 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, or more). In some embodiments, m is an integer of 1. In someembodiments, m is an integer ≥2 (e.g., 2, 3, 4, 6, 7, 8, 9, 10, 11, 12,or more). In some embodiments, m is an integer of 2, 3, or 4.

In some embodiments, the self-assembling peptide comprises or consistsof an amino acid sequence as set forth in [(Z)i(Y)j(X)k(Y)l]m (FormulaVIII), wherein each (X) is independently an ionic, polar amino acid,wherein each (Y) is independently a hydrophobic amino acid, wherein each(Z) is independently a non-ionic, polar amino acid, wherein each i, j,k, and l is independently an integer ≥1, wherein m is an integer ≥2, andwherein n=0 or an integer ≥1. In some embodiments, each i, j, k, and lis independently an integer of 1. In some embodiments, each i, j, k, andl is independently an integer of 2. In some embodiments, each i, j, k,and l is independently an integer of 3. In some embodiments, n is 0. Insome embodiments, n is an integer ≥1 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, or more). In some embodiments, m is an integer of 1. In someembodiments, m is an integer ≥2 (e.g., 2, 3, 4, 6, 7, 8, 9, 10, 11, 12,or more). In some embodiments, m is an integer of 2, 3, or 4.

In some embodiments, the self-assembling peptide comprises or consistsof an amino acid sequence as set forth in [(Y)i(Z)j(Y)k(X)l]m (FormulaVIII), wherein each (X) is independently an ionic, polar amino acid,wherein each (Y) is independently a hydrophobic amino acid, wherein each(Z) is independently a non-ionic, polar amino acid, wherein each i, j,k, and l is independently an integer ≥1, wherein m is an integer ≥2, andwherein n=0 or an integer ≥1. In some embodiments, each i, j, k, and lis independently an integer of 1. In some embodiments, each i, j, k, andl is independently an integer of 2. In some embodiments, each i, j, k,and l is independently an integer of 3. In some embodiments, n is 0. Insome embodiments, n is an integer ≥1 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, or more). In some embodiments, m is an integer of 1. In someembodiments, m is an integer ≥2 (e.g., 2, 3, 4, 6, 7, 8, 9, 10, 11, 12,or more). In some embodiments, m is an integer of 2, 3, or 4.

Each (X) in the self-assembling peptides of Formulas I-IV may be anacidic amino acid or a basic amino acid. For example, in someembodiments, each (X) is a basic amino acid, including but not limitedto arginine, lysine, histidine, and ornithine. In some embodiments, each(X) is an acidic amino acid, including, but not limited to aspartic acidand glutamic acid.

Each (Y) in the self-assembling peptides of Formulas I-IV may be analanine, a valine, a leucine, an isoleucine, a methionine, aphenylalanine, a tryptophan, or a glycine.

Each (Z) is selected from the group consisting of the group consistingof serine, threonine, tyrosine, cysteine, glutamine, asparagine, andmethionine.

The pharmaceutical compositions described herein may comprise one typeof self-assembling peptide or multiple different types ofself-assembling peptides. For example, in some embodiments, apharmaceutical composition provided herein may comprise one type ofself-assembling peptide (e.g., a self-assembling peptide comprising orconsisting of an amino acid sequence as set forth in Formula I). In someembodiments, the pharmaceutical compositions described herein comprisetwo or more types (e.g., two, three, four, five, six, seven eight, nine,ten, or more types) of self-assembling peptides. When a pharmaceuticalcomposition provided herein comprises more than one type ofself-assembling peptide, the self-assembling peptides may all compriseamino acid sequences as set forth in a single Formula (i.e., one ofFormulas I-IV) or may comprise amino acid sequences as set forth indifferent Formulas. When multiple types of self-assembling peptides arepresent in a pharmaceutical composition provided herein, the differenttypes of peptides can be capable of interacting and forming higher orderstructures (e.g., β-sheets).

In some embodiments, the self-assembling peptide comprises or consistsof between about 8 amino acid residues and about 50 amino acid residues.In some embodiments, the self-assembling peptide comprises or consistsof between about 8 amino acid residues and 18 amino acid residues. Forinstance, the self-assembling peptide may comprise 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,or 40 amino acid residues. The self-assembling peptide size may be suchthat a tertiary structure of the self-assembling peptide does notdisrupt the ability of the peptide to form a higher order structure(e.g., a nanofiber or a β-sheet) with other self-assembling peptides. Insome embodiments, the self-assembling peptides comprise or consist ofabout 12 amino acid residues. In some embodiments, the self-assemblingpeptides comprise or consist of about 13 amino acid residues. In someembodiments, the self-assembling peptides comprise or consist of about14 amino acid residues. In some embodiments, the self-assemblingpeptides comprise or consist of about 15 amino acid residues. In someembodiments, the self-assembling peptides comprise or consist of about16 amino acid residues. In some embodiments, the self-assemblingpeptides comprise or consist of about 17 amino acid residues. In someembodiments, the self-assembling peptides comprise or consist of about18 amino acid residues. In some embodiments, the self-assemblingpeptides comprise or consist of about 19 amino acid residues. In someembodiments, the self-assembling peptides comprise or consist of about20 amino acid residues. In some embodiments, the self-assemblingpeptides comprise or consist of about 21 amino acid residues. In someembodiments, the self-assembling peptides comprise or consist of about22 amino acid residues. In some embodiments, the self-assemblingpeptides comprise or consist of about 23 amino acid residues. In someembodiments, the self-assembling peptides comprise or consist of about24 amino acid residues. In some embodiments, the self-assemblingpeptides comprise or consist of about 25 amino acid residues. In someembodiments, the self-assembling peptides comprise or consist of about26 amino acid residues. In some embodiments, the self-assemblingpeptides comprise or consist of about 27 amino acid residues. In someembodiments, the self-assembling peptides comprise or consist of about28 amino acid residues. In some embodiments, the self-assemblingpeptides comprise or consist of about 29 amino acid residues. In someembodiments, the self-assembling peptides comprise or consist of about30 amino acid residues.

Exemplary self-assembling peptides are provided below in Table 1. Theexemplary self-assembling peptides of Table 1 include alternating ionic,polar amino acid residues, hydrophobic amino acid residues, andnon-ionic, polar amino acid residues. In some embodiments, thepharmaceutical compositions provided herein comprise a self-assemblingpeptide comprising or consisting of an amino acid sequence as set forthin SEQ ID NOs: 1-20 and 94-96.

TABLE 1 Exemplary self-assembling peptides SEQ ID NO: SequenceNumber of residues  1 KLNLKLNLKLNL 12  2 LNLKLNLKLNLK 12  3 NLKLNLKLNLKL12  4 LKLNLKLNLKLN 12  5 KLNLKLNLKLNLK 13  6 KLNLKLNLKLNLKLNLK 17  7IQIKIQIKIQIK 12  8 IQIKIQIKIQIKI 13  9 KIQIKIQIKIQIK 13 10 QIKIQIKIQIKIQ13 11 IKIQIKIQIKIQI 13 12 INIKINIKINIKI 13 13 NLELNLELNLEL 12 14NLDLNLDLNLDL 12 15 QLELQLELQLEL 12 16 LELQLELQLELQ 12 17 KANAKANAKANA 1218 KVNVKVNVKVNV 12 19 RANARANARANARANA 16 20 KLTLKLTLKLTL 12 94IEITIEITIEITI 13 95 ITIKITIKITIKI 13 96 KIQIKIQIKIQI 12

In some embodiments, the self-assembling peptides may comprise one ormore functional groups. In some embodiments, the functional groupprevents or delays the degradation of the self-assembling peptides. Insome embodiments, the self-assembling peptide comprises an N-terminalfunctional group. In some embodiments, the self-assembling peptidecomprises a C-terminal functional group. In some embodiments, theself-assembling peptide comprises both an N-terminal functional groupand a C-terminal functional group. For example, in some embodiments, thefunctional groups prevent or delay the degradation of theself-assembling peptides by enzymes (e.g., in vivo) (e.g., acetyl,formyl, chloroacetyl, benzyloxycarbonyl, bromoacetyl,tertbutoxycarbonyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl,methyl ester, benzyl ester, or t-butyl ester). Exemplary N-terminalfunctional groups include, but are not limited to, an acetyl, a formyl,pyroglutamyl (pGlu), biotin, polyethylene glycol (PEG), urea,alkylamine, a carbamate, a sulfonamide (e.g., 4-toluenesulfonyl,4-nitrobenzenesulfonyl), dansyl, 2,4-dintrophenyl, fluorescein,7-methoxycoumarin acetic acid, 9-fluorenylmethyloxycarbonyl, palmiticacid, succinyl, chloroacetyl, maleimide, benzyloxycarbonyl, bromoacetyl,nitrilotriacetyl, tertbutoxycarbonyl, 4-hydroxyphenylpropionic acid,allyloxycarbonyl, butyric acid, a fatty acid (e.g., hexanoic acid,octanoic acid, decanoic acid, palmitic acid, stearic acid, myristicacid, and lauric acid), and trityl. Exemplary C-terminal functionalgroups include, but are not limited to, an amido, an N-alkyl amide, analdehyde, an ester (e.g., a methyl ester, a benzyl ester, or a t-butylester), an alcohol, para-nitroanilide (pNA), 7-amino-4-methylcoumarin(Amc), a hydrazide, hydroxamic acid, chloromethylketone, p-nitroaniline,para-nitrophenol, hydroxysucinimide ester, fluoromethylketone,cysteamide, 9-fluorenemethyl (Fm) ester, allyl ester,2,4-dimethoxybenzyl ester, 2-phenylisopropyl ester, p-nitrobenzyl ester,and 2-chlorotrityl ester.

In some embodiments, the self-assembling peptides described herein cancomprise an N-terminal acetyl group and/or a C-terminal amine group. Forexample, exemplary self-assembling peptides comprising both of thesefunctional groups are provided below in Table 2. In some embodiments,the pharmaceutical compositions provided herein comprise aself-assembling peptide comprising or consisting of an amino acidsequence as set forth in SEQ ID NOs: 21-40 and 97-99.

TABLE 2 Exemplary self-assembling peptidescomprising an N-terminal acetyl group and a C-terminal amine group SEQNumber of ID NO: Name Sequence residues 21 KLNL12 Ac-KLNLKLNLKLNL-NH2 1222 LNLK12 Ac-LNLKLNLKLNLK-NH2 12 23 NLKL12 Ac-NLKLNLKLNLKL-NH2 12 24LKLN12 Ac-LKLNLKLNLKLN-NH2 12 25 KLNL13 Ac-KLNLKLNLKLNLK-NH2 13 26KLNL17 Ac-KLNLKLNLKLNLKLNLK-NH2 17 27 IQIK12 Ac-IQIKIQIKIQIK-NH2 12 28IQIK13 Ac-IQIKIQIKIQIKI-NH2 13 29 KIQI13 Ac-KIQIKIQIKIQIK-NH2 13 30QIKI13 Ac-QIKIQIKIQIKIQ-NH2 13 31 IKIQ13 Ac-IKIQIKIQIKIQI-NH2 13 32INIK13 Ac-INIKINIKINIKI-NH2 13 33 NLEL12 Ac-NLELNLELNLEL-NH2 12 34NLDL12 Ac-NLDLNLDLNLDL-NH2 12 35 QLEL12 Ac-QLELQLELQLEL-NH2 12 36 LELQ12Ac-LELQLELQLELQ-NH2 12 37 KANA12 Ac-KANAKANAKANA-NH2 12 38 KVNV12Ac-KVNVKVNVKVNV-NH2 12 39 RANA16 Ac-RANARANARANARANA-NH2 16 40 KLTL12Ac-KLTLKLTLKLTL-NH2 12 97 IEIT13 Ac-IEITIEITIEITI-NH2 13 98 ITIK13Ac-ITIKITIKITIKI-NH2 13 99 KIQI12 Ac-KIQIKIQIKIQI-NH2 12

In some embodiments, the self-assembling peptides described hereincomprise at least one biologically-active peptide motif. Without wishingto be bound by any particular theory, biologically-active peptide motifsmay facilitate one or more biological processes in, over or surroundinga pharmaceutical composition (e.g., a hydrogel) described herein,including, for example, cell adhesion, differentiation, proliferation,recruitment, and/or homing; neurite outgrowth; bioactive moleculerecruitment (e.g., as a binding site for the bioactive molecule),retention, and/or reconstitution. The biologically active peptide motifmay be present anywhere along the amino acid sequence of theself-assembling peptide and preferably does not interfere with theability of the self-assembling peptide to form a higher order structure(e.g., a nanofiber or a β-sheet) via intramolecular interactions orintermolecular interactions with other self-assembling peptides. Forexample, in some embodiments, the biologically-active peptide motif ispresent at the N-terminal end of the self-assembling peptide. In someembodiments, the biologically-active peptide motif is present at theC-terminal end of the self-assembling peptide. The self-assemblingpeptide can comprise one or more (e.g., one, two, three, four, five,six, seven, eight, or more) biologically active peptide motifs. When theself-assembling peptide comprises more than one biologically-activepeptide motifs, each motif may be of the same type or each motif may bedifferent. Exemplary biologically active peptide motifs may be derivedfrom proteins such as laminin-1, collagen IV, fibronectin, elastin, bonemarrow homing peptide 1, bone marrow homing peptide 2, or myelopeptide.Non-limiting examples of biologically active motifs are provided belowin Table 3. In some embodiments, a self-assembling peptide providedherein comprises a biologically active peptide motif comprising an aminoacid sequence as set forth in SEQ ID NOs:41-70.

TABLE 3 Exemplary biologically active peptide motifs. SEQ Peptide AminoID NO: Acid Sequence Protein 41 AASIKVAVSADR Laminin-1 42CSRARKQAASIKVAVSADR Laminin-1 43 YIGSR Laminin-1 44 PDGSR Laminin-1 45RYVVLPR Laminin-1 46 KAFDITYVRLKF Laminin-1 47 TAGSCLRKFSTM Collagen IV48 RNIAEIIKDI Laminin-1 49 YVRL Laminin-1 50 IRVTLN Laminin-1 51TTVKYIFR Laminin-1 52 SIKIRGTY Laminin-1 53 RQVFQVAYIIIKA Laminin-1 54FQIAYVIVKA Laminin-1 55 GQLFHVAYIIIKA Laminin-1 56 FHVAYVLIKA Laminin-157 LENGEIVSLVNGR Laminin-1 58 LGTIPG fibronectin 59 DGEA fibronectin 60REDV fibronectin 61 GVGVP elastin 62 GVGVAP elastin 63 IKVAV Laminin-164 PFSSTKT Bone marrow homing peptide 2 65 SKPPGTSS Bone marrow homingpeptide 1 66 SDPGYIGSR Laminin-1 67 RNIAELLKDI Laminin-1 68 PRGDSGYRGDSGCollagen IV 69 GFLGFPT myelopeptide 70 YGPDSGR Laminin-1

In some embodiments, the self-assembling peptide comprises abiologically active peptide motif shown below in Table 4. In someembodiments, the self-assembling peptide comprises or consists of anamino acid sequence as set forth in SEQ ID NOs:71-90.

TABLE 4 Exemplary self-assembling peptidescomprising biologically-active peptide motifs. SEQ Number ID of NO: NameAmino Acid Sequence residues 71 KLNL12YIG Ac-KLNLKLNLKLNLGYIGSR-NH2 1872 KLNL12PDG Ac-KLNLKLNLKLNLGPDGSR-NH2 18 73 KLNL12GVGAc-KLNLKLNLKLNLGGVGVAP-NH2 19 74 KLNL12PFS Ac-KLNLKLNLKLNLGPFSSTKT-NH220 75 KLNL12PRG Ac-KLNLKLNLKLNLGPRGDSGYRGDS 25 G-NH2 76 KIQI13YIGAc-KIQIKIQIKIQIKGYIGSR-NH2 19 77 KIQI13PDG Ac-KIQIKIQIKIQIKGPDGSR-NH2 1978 KIQI13GVG Ac-KIQIKIQIKIQIKGGVGVAP-NH2 20 79 KIQI13PFSAc-KIQIKIQIKIQIKGPFSSTKT-NH2 21 80 KIQI13PRG Ac-KIQIKIQIKIQIKGPRGDSGYRGD26 SG-NH2 81 NLEL12YIG Ac-NLELNLELNLELGYIGSR-NH2 18 82 NLEL12PDGAc-NLELNLELNLELGPDGSR-NH2 18 83 NLEL12GVG Ac-NLELNLELNLELGGVGVAP-NH2 1984 NLEL12PFS Ac-NLELNLELNLELGPFSSTKT-NH2 20 85 NLEL12PRGAc-NLELNLELNLELGPRGDSGYRGD 25 SG-NH2 86 RANA16YIGAc-RANARANARANARANAGYIGSR- 22 NH2 87 RANA16PDGAc-RANARANARANARANAGPDGSR- 22 NH2 88 RANA16GVGAc-RANARANARANARANAGGVGVAP- 23 NH2 89 RANA16PFSAc-RANARANARANARANAGPFSSTKT- 24 NH2 90 RANA16PRGAc-RANARANARANARANAGPRGDSGYR 29 GDSG-NH2

The concentration of self-assembling peptide present in thepharmaceutical compositions provided herein may be varied in order tochange the rheological properties of the composition. In someembodiments, the pharmaceutical compositions may comprise between about0.01% (w/v) and about 10% (w/v) of a self-assembling peptide describedherein. In some embodiments, the pharmaceutical compositions maycomprise between about 0.1% (w/v) and about 5% (w/v) of aself-assembling peptide described herein. In some embodiments, thepharmaceutical compositions may comprise between about 0.1% (w/v) andabout 2% (w/v) of a self-assembling peptide described herein. In someembodiments, the pharmaceutical compositions may comprise between about0.5% (w/v) and about 3% (w/v) of a self-assembling peptide describedherein. In some embodiments, the pharmaceutical compositions maycomprise between about 0.5% (w/v) and about 1.5% (w/v) of aself-assembling peptide described herein. In some embodiments, thepharmaceutical compositions may comprise between about 1% (w/v) andabout 3% (w/v) of a self-assembling peptide described herein. In someembodiments, the pharmaceutical compositions comprise about 0.5%, about1% (w/v), about 1.5% (w/v), about 2% (w/v), about 2.5% (w/v), about 3%(w/v), about 3.5% (w/v), about 4% (w/v), about 4.5% (w/v), or about 5%(w/v) of a self-assembling peptide described herein.

In some embodiments, the pharmaceutical compositions may be an aqueoussolution or a hydrogel, or it may be dehydrated (e.g., a powder).Hydrogels formed by the self-assembling peptides described herein may beporous or solid. In some embodiments, the hydrogels comprise poreshaving an average diameter of from about 1 nm to about 2,000 μm, of fromabout 10 nm to about 1,000 μm, of from about 10 nm to about 500 μm, offrom about 10 nm to about 100 μm, of from about 10 nm to about 1 μm, offrom about 5 nm to about 500 nm, or of from about 5 nm to about 250 nm.

In some embodiments, the pharmaceutical compositions provided herein maybe hydrogels of different sizes and geometric shapes, including filmsand particles, e.g., nanoparticles or microparticles. In someembodiments, the pharmaceutical composition may be layered onto asurface (e.g., a hydrogel). The pharmaceutical composition particle sizewill vary depending on the particular use intended for such a particle.In general, particles can have at least one dimension in the range fromabout 1000 μm to about 2000 μm (e.g., about 1,000 μm, about 1,100 μm,1,200 μm, 1,300 μm, 1,400 μm, 1,500 μm, 1,600 μm, 1,700 μm, 1,800 μm,1,900 μm, or 2,000 μm).

The self-assembling peptides described herein may be advantageouslyformulated at physiological pH and the self-assembling peptide(s)therein remain soluble and stable. In some embodiments, thepharmaceutical compositions described herein have a pH of from about 5to about 8. In some embodiments, the pharmaceutical compositions have apH of from about 6. to about 8. In some embodiments, the pharmaceuticalcompositions have a pH of from about 5.5. to about 7.5. In someembodiments, the pharmaceutical compositions have a pH of from about 6to about 7.4. In some embodiments, the pharmaceutical compositions havea pH of from about 6.5 to about 7.5. In some embodiments, thepharmaceutical compositions have a pH of from about 7 to about 7.5. Insome embodiments, the pharmaceutical compositions have a pH of about6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1,about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about7.8, about 7.9, or about 8.0. The pH of a pharmaceutical compositiondescribed herein may be regulated by including a buffer system.Alternatively, the self-assembling peptides may act as a buffer in thepharmaceutical compositions. The pH of the pharmaceutical compositionmay be adjusted using one or more acids or bases such as sodiumhydroxide, potassium hydroxide, hydrochloric acid, phosphoric acid,sodium carbonate and sodium hydrogen carbonate.

In some embodiments, the pharmaceutical compositions may comprise abuffer such as phosphate buffered saline (PBS). In some embodiments, thepharmaceutical compositions may comprise a medium for cell culture,including, but not limited to Dulbecco's Modified Eagle's Medium (DMEM),DME/F12, Minimal Essential Medium (MEM), Basal Medium Eagle (BME), RPMI1640, F-10, F-12, α-Minimal Essential Medium (α-MEM), Glasgow's MinimalEssential Medium (G-MEM), PF CHO (SAFC Biosciences) and Iscove'sModified Dulbecco's Medium.

The pharmaceutical compositions described herein can be formulated suchthat the pH of the composition is neutral or physiological (e.g., a pHof from about 6 to about 8) and the self-assembling peptides thereinhave a non-zero net charge. Without wishing to be bound by anyparticular theory, when formulated at neutral or physiological pH, thenon-zero net charge of the self-assembling peptides enables the peptidesto readily form electrostatic interactions with molecules of theopposite charge. This property can be advantageously used to promoteadhesion of the pharmaceutical composition to a desired surface. This isparticularly advantageous in clinical applications where thepharmaceutical compositions are applied to tissue enriched withbiomolecules (e.g., glycoproteins) having a non-zero net charge.

Moreover, without wishing to be bound by any particular theory,self-assembling peptides described herein having a net positive chargewhen formulated at physiological pH, are particularly advantageous intherapeutic applications as they may be bactericidal and/orbacteriostatic. Thus, the self-assembling peptides may be readily usedin a variety of applications where reduced bacterial burden is desired.

In some embodiments, the net charge of the self-assembling peptide isgreater than or equal to +1 or less than or equal to −1 when thepharmaceutical composition has a pH of from about 5 to about 8. In someembodiments, the net charge of the self-assembling peptide is from about+1 to about +6 (e.g., +1, +2, +3, +4, +5, or +6) when the pharmaceuticalcomposition has a pH of from about 5 to about 8 (e.g., 7.4). In someembodiments, the net charge of the self-assembling peptide is from about−1 to about −6 (e.g., −1, −2, −3, −4, −5, or −6) when the pharmaceuticalcomposition has a pH of from about 5 to about 8 (e.g., 7.4).

In some embodiments, the pharmaceutical compositions provided hereinhave an elastic modulus in the range between about 10 Pascal (Pa) toabout 10,000 Pa. As used herein, the term “elastic modulus” refers to acomposition's tendency to be deformed elastically (i.e.,non-permanently) when a force is applied to it. Generally, the elasticmodulus of an object is defined as the slope of its stress-strain curvein the elastic deformation region. Specifying how stress and strain areto be measured, including directions, allows for many types of elasticmoduli to be defined. Young's modulus (E) describes tensile elasticity,or the tendency of an object to deform along an axis when opposingforces are applied along that axis; it is defined as the ratio oftensile stress to tensile strain. It is often referred to simply as theelastic modulus. The shear modulus or modulus of rigidity (G or μ)describes an object's tendency to shear (the deformation of shape atconstant volume) when acted upon by opposing forces; it is defined asshear stress over shear strain. The shear modulus is part of thederivation of viscosity. The bulk modulus (K) describes volumetricelasticity, or the tendency of an object to deform in all directionswhen uniformly loaded in all directions; it is defined as volumetricstress over volumetric strain and is the inverse of compressibility. Thebulk modulus is an extension of Young's modulus to three dimensions. Insome embodiments, a pharmaceutical composition described herein (e.g., ahydrogel) has a storage modulus of at least about 10 Pa. In someembodiments, a pharmaceutical composition described herein has a storagemodulus of at least about 50 Pa. In some embodiments, a pharmaceuticalcomposition described herein has a storage modulus of at least about 100Pa. In some embodiments, a pharmaceutical composition described hereinhas a storage modulus of at least about 500 Pa. In some embodiments, apharmaceutical composition described herein has a storage modulus of atleast about 1,000 Pa. In some embodiments, a pharmaceutical compositiondescribed herein has a storage modulus of between about 10 Pa and about5,000 Pa. In some embodiments, a pharmaceutical composition describedherein has a storage modulus of between about 50 Pa and about 500 Pa. Insome embodiments, a pharmaceutical composition described herein has astorage modulus of between about 10 Pa and about 1,000 Pa. In someembodiments, a pharmaceutical composition described herein has a storagemodulus of between about 500 Pa and about 5,000 Pa.

In some embodiments, increased ionic strength may increase the stiffnessand/or gelation kinetics of the self-assembling peptides present in thepharmaceutical compositions described herein. In some embodiments,increased ionic strength may be physiological ionic strength. The ionicstrength of the pharmaceutical compositions may be increased when thecompositions are administered into and/or onto a subject (e.g., a humansubject). Alternatively, the ionic strength of the pharmaceuticalcompositions described herein may be increased by admixing thecompositions with one or more tonicity agents. Exemplary tonicity agentsinclude, but are not limited to, sodium chloride, calcium chloride,potassium chloride, and potassium phosphate. In some embodiments, thepharmaceutical compositions described herein do not include a tonicityagent. In some embodiments, the pharmaceutical compositions describedherein comprise between about 0.01 M to about 0.3 M tonicity agent. Insome embodiments, the pharmaceutical compositions described hereincomprise between about 0.1 M to about 0.3 M tonicity agent. In someembodiments, the pharmaceutical compositions described herein comprisebetween about 0.1M to about 0.2 M tonicity agent. In some embodiments,the pharmaceutical compositions described herein comprise 0.01 M, 0.02M, 0.03 M, 0.04 M, 0.05 M, 0.06 M, 0.07 M, 0.08 M, 0.09 M, 0.1 M, 0.15M, 0.2 M, 0.25 M, or 0.3 M tonicity agent.

In some embodiments, the pharmaceutical compositions described hereinare formulated such that they are hypoosmotic in relation to the site ofadministration in a subject. In some embodiments, the pharmaceuticalcompositions described herein are formulated such that they areisoosmotic in relation to the site of administration in a subject. Insome embodiments, the pharmaceutical compositions described herein areformulated such that they are hyperosmotic in relation to the site ofadministration in a subject.

In some embodiments, the pharmaceutical compositions described hereincomprise a cell (e.g., an isolated cell). In some embodiments, thepharmaceutical composition (e.g., a hydrogel) can enhance the viabilityof a cell therein thereby facilitating the delivery of viable cells to adesired site (e.g., an injured or defective body tissue). In someembodiments, the pharmaceutical composition is an aqueous solution thatcomprises between about 10⁴ to about 10⁸ cells/mL (e.g., 10⁴, 10⁵, 10⁶,10⁷ cells/mL). In some embodiments, the pharmaceutical composition is anaqueous solution that comprises between about 10⁴ to about 10⁶ cells/mL.In some embodiments, the pharmaceutical composition comprises one celltype. In some embodiments, the pharmaceutical composition comprises morethan one cell type (e.g., two, three, four, five, or more cell types).

Any appropriate cell may be comprised in the pharmaceuticalcompositions, depending on the desired application. For example, in someembodiments the pharmaceutical composition (e.g., a hydrogel) is seededwith an animal cell or a plant cell. When a hydrogel, the cells may beencapsulated within the hydrogel. In some embodiments, thepharmaceutical composition comprises a mammalian cell. Exemplarymammalian cells, include, but are not limited to, stem cells (embryonicstem cells, mesenchymal stem cells, bone-marrow derived stem cells,hematopoietic stem cells, neural stem cells, and hair follicular stemcells), chondrocyte progenitor cells, pancreatic progenitor cells,myoblasts, fibroblasts, keratinocytes, neuronal cells, glial cells,astrocytes, pre-adipocytes, adipocytes, vascular endothelial cells,endothelial progenitor cells, mesenchymal cells, neural stem cells,immune cells, (e.g., B-cells and T-cells), smooth muscle progenitorcells, cardiac myocytes, fetal dermal fibroblasts, epidermalkeratinocytes, myoblasts, and capillary endothelial cells. In someembodiments, the cell is a genetically-modified cell (e.g., a cellmodified to express and secrete a desired compound such as a growthfactor, a differentiation factor, a cytokine, a chimeric antigenreceptor, or an antibody or a fragment thereof). In some embodiments,the cell is a tissue culture cell line cell. Exemplary tissue culturecell line cells include, but are not limited to, C166 cells, C6 gliomacell line, AML12, HeLa cells, and Chinese Hamster Ovary cells (CHOcells).

In some embodiments, the pharmaceutical compositions provided hereincomprise a bioactive molecule, including but not limited to, anextracellular matrix protein (e.g., fibronectin, vitronectin, andlaminin), a cytokine, a growth factor, a differentiation factor,insulin, a nucleic acid, a vitamin, a fatty acid, and a therapeuticagent.

Exemplary growth factors and cytokines include, but are not limited to,stem cell factor (SCF), granulocyte-colony stimulating factor (G-CSF),granulocyte-macrophage stimulating factor (GM-CSF), stromal cell-derivedfactor-1, vascular endothelial growth factor (VEGF), platelet derivedgrowth factor (PDGF), an angiopoeitin, epidermal growth factor (EGF),basic fibroblast growth factor (bFGF), hepatocyte nuclear factor-1(HNF-1), nerve growth factor (NGF), bone morphogenetic protein (BMP),fibroblast growth factor (FGF), hepatocyte growth factor, insulin-likegrowth factor (IGF-1), interleukin (IL)-3, IL-la, IL-43, IL-6, IL-7,IL-8, IL-11, and IL-13, colony-stimulating factors, thrombopoietin,erythropoietin, fit3-ligand, and tumor necrosis factor α (TNFα).

In some embodiments, the bioactive molecule is a therapeutic agent. Asused herein, the term “therapeutic agent” refers to a compound used inthe diagnosis, treatment, or prevention of a disease or disorder in asubject. Any therapeutic agent known to be of benefit in the diagnosis,treatment or prevention of a disease or disorder may be included in apharmaceutical compositions provided herein. Therapeutic agents includepharmacologically active compounds (e.g., antibiotics andanti-inflammatory agents), hormones, DNA (e.g., plasmid DNA), RNA,siRNA, shRNA, anti-sense RNA, proteins (e.g., antibodies and fragmentsthereof, and enzymes), lipids, pro-inflammatory molecules, andcombinations thereof. Any of the therapeutic agents may be combined tothe extent such combination is biologically compatible. The amount oftherapeutic agent in the pharmaceutical composition will depend onvarious factors including, for example, the effective dose oftherapeutic agent required for a particular course of action and therequired period of time for release of the therapeutic agent. Additionalexemplary therapeutic agents and appropriate dosing amounts and regimensare described, for example, in Harrison's Principles of InternalMedicine, 19th Edition, Eds. D. L. Kasper et al., McGraw-Hill, New York,N.Y. (2015); Physician's Desk Reference, 71st Edition, Montvale, N.J.,Physician's Desk Reference Inc. (2016); and Goodman and Gilman's: ThePharmacological Basis of Therapeutics, 13th Edition, Eds. L. L. Bruntonet al., McGraw-Hill, New York, N.Y. (2018); each of which isincorporated herein by reference.

Uses of the Self-Assembling Peptides and Pharmaceutical Compositions

The self-assembling peptides and pharmaceutical compositions describedherein may be used in a variety of in vitro and in vivo applications,including cell culture applications and clinical applications.

Cell Culture

In some embodiments, the self-assembling peptides and pharmaceuticalcompositions described herein may be used in the culture of cells (e.g.,isolated human cells). For instance, hydrogels comprising theself-assembling peptides described herein may be substrates for cellculture. As described above, hydrogels formed by the self-assemblingpeptides described herein may comprise one or higher order structures(e.g., nanofibers and three-dimensional mesh) that provide aspatiotemporal substrate for cells. Advantageously, since the hydrogelscan be formulated at physiological pH, they may provide an adequateenvironment for cell growth and proliferation.

Any of the cell types described in detail above may be cultured in thehydrogels described herein. Cells may be seeded directly onto apreformed hydrogel or they may be admixed with an aqueous pharmaceuticalcomposition, which upon gelation forms a hydrogel. The liquid phase ofthe hydrogel may be supplemented with one or more culture mediumcomponents (e.g., growth factors and serum) to provide the necessaryfactors to promote cell survival and proliferation. Appropriateconditions and factors necessary to sustain cell survival and growth areknown in the art (see, e.g., Freshney, Culture of Animal Cells: A Manualof Basic Technique, Wiley-Liss, New York, N.Y. (2000); and Cells: ALaboratory Manual (Spector, D. L., Goldman, R. D. and Leinwand, L. A.,eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(1998).

The self-assembling peptides and pharmaceutical compositions providedherein may be used in a variety of applications including, for example,to perform cell culture, in cosmetics (e.g., skin and hair careproducts), for tissue engineering, as coating agents (e.g., for medicaldevices such as contact lenses), as lubricants (e.g., in ophthalmicsolutions or as joint lubricants), as hemostatic agents, as desiccants,as bone filler material, as artificial vitreous bodies, as artificiallenses, as drug delivery devices, as water retention materials, and inbiomaterial applications.

Clinical Applications

In some embodiments, the self-assembling peptides and pharmaceuticalcompositions described herein may be used in a variety of clinicalapplications. Thus, provided herein are various methods of treating asubject by administering an effective amount of a pharmaceuticalcomposition described herein to a specific site in or on a subject. Asdescribed above, a particular advantage of the pharmaceuticalcompositions described herein is that they may be formulated atphysiological pH (e.g., from about pH 6 to about pH 8), and thereforemay result in reduced pain and reduced tissue damage upon contact withthe subject, as compared to pharmaceutical compositions ofself-assembling peptides that are formulated at acidic or basic pH.

The amount, for example, volume or concentration, of pharmaceuticalcompositions that is administered (for example, applied or injected) toa subject may vary depending upon the form of the pharmaceuticalcomposition (e.g., as an aqueous solution or hydrogel) and the route ofadministration utilized. The exact formulation, route of administration,volume, and amount of pharmaceutical composition can be chosen in viewof the subject's condition and in view of the particular target area orlocation to which the pharmaceutical composition is administered.Specific dosage and treatment regimens for any particular subject maydepend upon a variety of factors, including the specific self-assemblingpeptide, the dimension of the area that is being treated, the desiredthickness of the resulting hydrogel (when an aqueous solution isadministered), and the length of time of treatment. Other factors thatmay affect the specific dosage and treatment regimens include the age,body weight, health status, sex, time of administration, rate ofdegradation, the severity and course of the disease, condition orsymptoms. In some embodiments, the pharmaceutical composition may beadministered in a single dose. In other embodiments, the pharmaceuticalcomposition may be administered in more than one dose (e.g., two, three,four, five, six, or more doses).

In some embodiments, a pharmaceutical composition in the form of apre-polymerized hydrogel is administered to a subject. In someembodiments, the pharmaceutical composition is a hydrogel that is formedin vitro and administered to the desired location in the subject.

In some embodiments, a pharmaceutical composition in the form of anaqueous solution is administered to a subject. A hydrogel may be formedin vivo after administration of the aqueous solution. Since theself-assembling peptides polymerize in response to changes in tonicity,the hydrogel may form upon contact with a subject during theadministration or shortly thereafter.

The pharmaceutical compositions may be administered by introducing adelivery device at or near a predetermined or desired target area of asubject. The delivery device may be a conventional device or designed toaccomplish at least one of to reach a specific target area, achieve aspecific dosing regimen, deliver a specific target volume, amount, orconcentration, and deliver accurately to a target area. Suitabledelivery devices include, but are not limited to, syringes, pipettes,tubes, catheters, syringe catheters, auto-injectors, and otherneedle-based device to the predetermined or desired target area of asubject. The gauge of a syringe needle may be selected to provide anadequate flow of a pharmaceutical composition from the syringe to thetarget area.

The effective amount may comprise volumes of from about 0.1 milliliters(mL) to about 100 mL of a pharmaceutical composition (if administered asan aqueous solution). In some embodiments, an effective amount may befrom about 0.1 mL to about 10 mL of pharmaceutical composition). In someembodiments, an effective amount may be from about 0.5 mL to about 5 mLof pharmaceutical composition. In some embodiments, an effective amountmay be from about 1 mL to about 5 mL of pharmaceutical composition. Insome embodiments, an effective amount may be about 0.5 mL ofpharmaceutical composition. In some embodiments, an effective amount maybe about 1.0 mL of pharmaceutical composition. In some embodiments, aneffective amount may be about 1.5 mL of pharmaceutical composition. Insome embodiments, an effective amount may be about 2.0 mL ofpharmaceutical composition. In some embodiments, an effective amount maybe about 2.5 mL of pharmaceutical composition. In some embodiments, aneffective amount may be about 0.1 mL to about 5 mL per cm² of targetarea. In some embodiments, an effective amount may be about 1 mL per cm²of target area. In some embodiments, an effective amount may be about 2mL per cm² of target area.

The pharmaceutical compositions may be administered to a subject by anyappropriate route known in the art including, without limitation,injection, implantation, microinjection, and direct application.Administration via injection includes, without limitation, cutaneous,intramuscular, intradermal, subdermal, and subcutaneous injections.

In some embodiments, the pharmaceutical composition is administered(e.g., injected or implanted) to a subject in a single dose at onelocation to produce the desired result. In some embodiments, thepharmaceutical composition is administered as several doses to producethe desired result. In some embodiments, when multiple doses of thepharmaceutical composition are administered, each administration occursafter a specific period of time, e.g., 1 day, 2 days, 3 days, 4 days, 5days, 6 days, 1 week, 2 weeks, 1 month, 2 months, 3 months, or more. Insome embodiments, a hydrogel that has formed or is present at a site ina subject may be removed by contacting the hydrogel with a solutionincluding a hypotonic concentration of tonicity agent as compared to thehydrogel. In some embodiments, a hydrogel that has formed or is presentat a site in a subject may be removed by contacting the hydrogel withwater. In some embodiments, a hydrogel that has formed or is present ata site in a subject may be removed by contacting the hydrogel with asolution that is hypotonic in relation to the site in the subject. Insome embodiments, a hydrogel that has formed or is present at a site ina subject may be removed by contacting the hydrogels with a solutionthat is isotonic with sugars (e.g., dextrose and sucrose) to the site inthe subject. In some embodiments, a hydrogel that has formed or ispresent at a site in a subject may be removed by contacting thehydrogels with a solution that is isotonic with salts bearing low ionicstrength (e.g., NaCl—“Normal Saline”) compared to the salts in bodyfluid.

The hydrogel may be contacted with the solution for a sufficient periodof time to disrupt (e.g., disrupt the structure or morphology) of thehydrogel and/or cause the hydrogel to detach from the site in thesubject. In some embodiments, the hydrogel is contacted with thesolution for about 5 minutes, for about 10 minutes, for about 20minutes, for about 30 minutes, for about 1 hour, for about 2 hours, forabout 3 hours, or more.

In some embodiments, a pharmaceutical composition described herein maybe used as a delivery device to specifically target release of abioactive molecule or therapeutic agent. In some embodiments, thepharmaceutical composition may be formulated such that the compositionallows for the spontaneous release of a bioactive molecule ortherapeutic agent (e.g., after administration to a subject). In someembodiments, the pharmaceutical composition may be formulated such thatthe pharmaceutical composition allows for the controlled release of abioactive molecule or therapeutic agent. In some embodiments, thebioactive molecule or therapeutic agent is released from thepharmaceutical composition (e.g., a hydrogel) for a prolonged period oftime (e.g., from about 12 hours to about 2 months). In some embodiments,the bioactive molecule or therapeutic agent is released from thepharmaceutical composition for about 12 hours, one day, two days, threedays, four days, five days, six days, one week, two weeks, three weeks,one month, two months, three months, or more. The release kinetics willvary according to the amino acid sequence of the self-assembling peptidepresent in the pharmaceutical composition, the concentration of theself-assembling peptide in the pharmaceutical composition, and thebiochemical and physical properties of the bioactive molecule ortherapeutic agent.

1. Gastrointestinal Obstruction

The self-assembling peptides and pharmaceutical compositions describedherein may be used to prevent gastrointestinal obstruction in a subject.Methods of preventing gastrointestinal obstruction using apharmaceutical composition comprising self-assembling peptides are knownin the art and can be adapted for delivering the pharmaceuticalcompositions and self-assembling peptides described herein (see, e.g.,U.S. Pat. No. 9,724,448, the entire contents of which are expresslyincorporated herein by reference). For example, endoscopic mucosalresection (EMR) and endoscopic submucosal dissection (ESD) are primarysurgical options for the resection of lesions, such as polyps, ulcers,and cancerous tumors in the gastrointestinal tract (see, e.g., Wallace(2017) Gastroenterol. Hepatol. (NY) 13(6): 371-4). Although EMR and ESDare minimally invasive procedures, obstructions in the gastrointestinaltract can be caused by scar contraction/shrinking during the healingprocess. One form of gastrointestinal obstruction may be a stenosis, anarrowing in a tubular organ or structure, such as the gastrointestinaltract, which may lead to a partial or full obstruction in thegastrointestinal tract. Methods of preventing stenosis in thegastrointestinal tract of a subject (e.g., in one or more of the mouth,throat, esophagus, stomach, small intestine, large intestine, colon orrectum) are provided. In some embodiments, an effective amount of apharmaceutical compositions (e.g., an aqueous solution) described hereinare administered using a medical device (e.g., a syringe, pipette, tube,syringe catheter, catheter, or endoscope) to a site in thegastrointestinal tract of the subject. In some embodiments, the aqueoussolution forms a hydrogel at the site of administration thereby allowingfor the prevention of stenosis at the site of administration. In someembodiments, the self-assembling peptides may facilitate mucosalepithelium formation to prevent or reduce post-operative scar formation,which may contribute to the prevention or reduction in gastrointestinalobstruction or stenosis. In some embodiments, the hydrogel provides ascaffold for the infiltration of cells that promote healing of the siteof administration of the hydrogel.

In some embodiments, a pharmaceutical composition provided herein (e.g.,an aqueous solution) may be administered (e.g., by injection) tosubmucosa at a site in the gastrointestinal tract of a subject prior toperforming an excision of a lesion (e.g., using EMR or ESD). Thesubmucosa is a thin connective tissue layer with a lax structure. Insome embodiments, injection of the pharmaceutical composition to thesubmucosa forms a bulla and lifts the lesion above, thereby facilitatingremoval of the lesion. In some embodiments, the pharmaceuticalcomposition elevates the submucosa for a period of time from about 20minutes to about 1 hour. In some embodiments, the pharmaceuticalcomposition elevates the submucosa for about 20 minutes, about 30minutes, about 40 minutes, about 50 minutes, about 1 hour, about 2hours, about three hours, about four hours, about five hours, or more.

2. Tissue Repair or Regeneration

The self-assembling peptides and pharmaceutical compositions describedherein may be used to promote or enhance the repair or regeneration oftissue in a subject. The self-assembling peptides and pharmaceuticalcompositions may promote or enhance distinct types of tissue including,but not limited to, skin, bone, cartilage, neural tissue, ligaments,tendons, vascular tissue, and muscle (e.g. cardiac tissue). In someembodiments, the subject has a congenital disease or condition resultingin a need for tissue repair or regeneration. In some embodiments, thesubject has experienced an injury resulting in a need for tissueregeneration. The injury may occur as a result of surgery, trauma,stroke, tumor, a disease or disorder (e.g., a neurodegenerative diseaseor disorder). The methods and compositions described herein may restorethe structural and/or functional integrity of the tissue (e.g., to thestructural and/or functional state of the tissue prior to an injury).Methods for promoting tissue regeneration using hydrogels comprisingself-assembling peptides are known in the art and can be adapted for usewith the self-assembling peptides and hydrogels described herein (see,e.g., U.S. Pat. No. 7,846,891 and U.S. Publication Nos. 2016/0362451 and2017/0128172, the entire contents of each which are expresslyincorporated herein by reference).

In some embodiments, the self-assembling peptides and pharmaceuticalcompositions described herein may be used to ameliorate or treat theeffects of tissue degeneration of an organ, to repair an injury to anorgan or other body structure or to form an organ or other bodystructure. Such organs or body structures include, but are notnecessarily limited to, vascular tissue, brain, nervous tissue,esophagus, fallopian tube, heart, intestines, gallbladder, kidney,liver, lung, ovaries, pancreas, prostate, bladder, bone, spinal cord,spleen, stomach, testes, thymus, thyroid, trachea, ureter, urethra,uterus, and skin.

The repair and regeneration of tissue can be enhanced by supplyingbioactive molecules such as growth factors, cell adhesion molecules,integrins, etc. with the self-assembling peptides and pharmaceuticalcompositions. In some embodiments, the bioactive molecules are presentin the pharmaceutical composition. In some embodiments, cells thatproduce one or more bioactive molecules are present in thepharmaceutical composition. For example, genetically-modified, cellsthat produce and/or secrete one or more bioactive molecules may bepresent in the pharmaceutical composition.

The self-assembling peptides and pharmaceutical compositions describedherein may be used to promote the regeneration or repair of oculartissue (e.g., an optic nerve, corneal stromal layer and lens cortex).For example, the self-assembling peptides and pharmaceuticalcompositions described herein may be used to treat a subject having aretinopathy or a retinal/macular disorder.

In some embodiments, the self-assembling peptides and pharmaceuticalcompositions described herein may be used to promote or enhance repairor regeneration of neural tissue at a site of injury in a subject. Forexample, when administered to a site of injury, the pharmaceuticalcompositions described herein provide an environment that is permissivefor the repair or regeneration of neural tissue and axon growth at thesite of injury.

3. Wound Healing and Anti-Microbial Dressing

The self-assembling peptides and pharmaceutical compositions describedherein may be used to promote wound healing or skin reconstruction ortreat a wound (e.g., a burn) in a subject in need thereof. For example,the pharmaceutical compositions described herein may be directly appliedto a wound or may be adapted for use with a gauze or a sheet in order topromote healing of the wound, promote skin reconstruction, or treat thewound. For example, in some embodiments, the pharmaceutical compositionsdescribed herein may be injected into biopsy sites or wound sitescreated by a surgical intervention (e.g., removal of a tumor). Thepharmaceutical compositions described herein may also be used tofacilitate healing in chronic wounds, such as skin lesions and diabeticulcers. Methods for promoting wound healing or skin reconstruction andfor treating wounds using self-assembling peptides are known in the artand can be adapted for use with the self-assembling peptides andpharmaceutical compositions described herein (see, e.g., U.S.Publication No. 2011/0002880, and International Publication No. WO2017/210416, the entire contents of each of which are expresslyincorporated herein by reference).

In some embodiments, the self-assembling peptides and pharmaceuticalcompositions described herein may be used as anti-microbial agents. Insome embodiments, the self-assembling peptides and pharmaceuticalcompositions described herein may be used as an anti-microbial dressing.

4. Hemostasis

The self-assembling peptides and pharmaceutical compositions describedherein may be used to promoted hemostasis in a subject. Theself-assembling peptides and pharmaceutical compositions describedherein may be used to stop or control blood loss from vessels (e.g., anartery, a vein, an aorta) and organs of the body of a subject (e.g.,during surgery or after a traumatic injury). Methods for promotinghemostasis in a subject are known in the art and may be adapted for usewith the self-assembling peptides and pharmaceutical compositionsdescribed herein (see, e.g., International Publication No.WO2017/210421, the entire contents of which are expressly incorporatedherein by reference).

In some embodiments, the self-assembling peptides and pharmaceuticalcompositions described herein may be used to create a physical barrierto stop or prevent bleeding during a surgical or endoscopic procedures(e.g., during a hepatectomy, a splenectomy, a vaginoplasty, acholecystectomy, a coronary bypass, or a femoral bypass). In someembodiments, the self-assembling peptides and pharmaceuticalcompositions described herein may be used to stop or prevent exudativehemorrhage from blood vessels and parenchyma of solid organs. In someembodiments, the self-assembling peptides and pharmaceuticalcompositions described herein may be used to stop or prevent exudativehemorrhage from vascular anastomosis (e.g. anastomosis to a native orartificial vessel). In some embodiments, the self-assembling peptidesand pharmaceutical compositions described herein may be used to stop orprevent exudative hemorrhage from a small vessel or capillary vessel ofthe gastrointestinal tract of a subject (e.g., during an endoscopicsub-mucosal dissection (EMD) of the gastrointestinal tract, alaparoscopic resection of the gastrointestinal tract, or an endoscopicmucosal resection (EMR) of the gastrointestinal tract.

5. Local Drug/Therapeutic Agent Delivery

In one aspect, the present disclosure provides a combination compositioncomprising (i) self-assembling peptides comprising a self-assemblingpeptide or pharmaceutical composition described herein, and (ii) one ormore payload agents (e.g., therapeutic agents), wherein a combinationcomposition has a storage modulus of about 0.1 to about 100 Pa (e.g., at5 rad/sec of frequency and 0.1 Pa of oscillation stress), and/or thecombination composition has a viscosity in the range of about 0.5 Pa toabout 50,000 Pa at room temperature. In some embodiments, one or morepayload agents (e.g., therapeutic agents) of a combination compositionare distributed substantially homogeneously within the combinationcomposition.

In another aspect, the self-assembling peptides and pharmaceuticalcompositions described herein may be used for local delivery of drugsand/or therapeutic agents to diseased and/or defected sites in the body.Drugs/therapeutics can be mixed with peptide solution and injected usinga needle, nozzle, or catheter. Drugs can be delivered and localized inthe tissue, and then can be slowly released before hydrogel is absorbedin the body.

The term “agent” as used herein refers to a compound or entity of anychemical class including, for example, polypeptides, nucleic acids,saccharides, lipids, small molecules, metals, or combinations thereof.In some embodiments, an agent is or comprises a natural product in thatit is found in and/or is obtained from nature. In some embodiments, anagent is or comprises one or more entities that are man-made in that itis designed, engineered, and/or produced through action of the hand ofman and/or is not found in nature. In some embodiments, an agent may beutilized in isolated or pure form; in some embodiments, an agent may beutilized in crude form. In some embodiments, potential agents areprovided as collections or libraries, for example that may be screenedto identify or characterize active agents within them. Some particularembodiments of agents that may be utilized in accordance with thepresent invention comprise small molecules, antibodies, antibodyfragments, aptamers, nucleic acids (e.g., siRNAs, shRNAs, DNA/RNAhybrids, antisense oligonucleotides, mRNA, CRISPR systems, andribozymes), peptides, peptide mimetics, etc. In some embodiments, theagent is or comprises a polymer. In some embodiments, the agent is not apolymer and/or is substantially free of any polymer. In someembodiments, the agent contains at least one polymeric moiety. In someembodiments, the agent lacks or is substantially free of any polymericmoiety. In some embodiments, the agent is a cell and/or tissue. In someembodiments, the agent is or comprises a cellular lysate. In someembodiments, the agent is or comprises cellular material and/ormulti-cellular material (e.g., micro-column grafts and/or micro-grafts).

As used herein, the phrase “therapeutic agent” in general refers to anyagent that elicits a desired pharmacological effect when administered toan organism. In some embodiments, an agent is considered to be atherapeutic agent if it demonstrates a statistically significant effectacross an appropriate population. In some embodiments, the appropriatepopulation may be a population of model organisms. In some embodiments,an appropriate population may be defined by various criteria, such as acertain age group, gender, genetic background, preexisting clinicalconditions, etc. In some embodiments, a therapeutic agent is a substancethat can be used to alleviate, ameliorate, relieve, inhibit, prevent,delay onset of, reduce severity of, and/or reduce incidence of one ormore symptoms or features of a disease, disorder, and/or condition. Insome embodiments, a “therapeutic agent” is an agent that has been or isrequired to be approved by a government agency before it can be marketedfor administration to humans. In some embodiments, a “therapeutic agent”is an agent for which a medical prescription is required foradministration to humans.

In some particular embodiments, combination compositions of theinvention comprise one or more payload agents, e.g., therapeutic agentsor detection agents. Such agents comprise, e.g., a compound or entity ofany chemical class including, for example, polypeptides, nucleic acids,saccharides, lipids, small molecules, metals, or combinations thereof.As will be clear from context, in some embodiments, an agent can be orcomprise a cell or organism, or a fraction, extract, or componentthereof. In some embodiments, an agent is or comprises a natural productin that it is found in and/or is obtained from nature. In someembodiments, an agent is or comprises one or more entities that areman-made in that it is designed, engineered, and/or produced throughaction of the hand of man and/or is not found in nature. In someembodiments, an agent may be utilized in isolated or pure form; in someembodiments, an agent may be utilized in crude form. In someembodiments, potential agents are provided as collections or libraries,for example that may be screened to identify or characterize activeagents within them. Some particular embodiments of agents that may beutilized in accordance with the present invention comprise smallmolecules (e.g. antibiotics, anticancer drugs, antipain drugs,antiinflammatory drugs, steroids, anti-psychotics), antibodies, antibodyfragments, aptamers, nucleic acids (e.g., siRNAs, shRNAs, DNA/RNAhybrids, antisense oligonucleotides, ribozymes), peptides, peptidemimetics, proteins, fusion proteins, vaccines, anti-coagulants,cytokines, hormones, enzymes, blood factors, extracellular matrixcomponents, etc. In some embodiments, an agent may be selected from thegroup consisting of micrograft tissue, drugs (e.g., antibiotics), andbiologies (e.g., growth factors and/or other molecules/proteins). Insome embodiments, an agent is a cytokine (e.g., epidermal growth factor,nerve growth factor, transforming growth factor-alpha and beta,platelet-derived growth factor, insulin-like growth factor, vascularendothelial growth factor).

In some embodiments, a self-assembling peptide or pharmaceuticalcomposition described herein combined with one or more payload agents(e.g., therapeutic agents) is used to treat a disease or disorder, e.g.,a disease or disorder known to be or suspected to be treated by atherapeutic agent described herein (e.g., infection, cancer,cardiovascular disease, neurological disease), and/or used inwound-healing, bone/cartilage repair/regeneration, soft tissueregeneration. A self-assembling peptide or pharmaceutical compositiondescribed herein combined with one or more payload agents (e.g.,therapeutic agents) can be administered to a subject in a variety ofways, and administration is not limited to any particular method. Insome embodiments, a self-assembling peptide or pharmaceuticalcomposition described herein combined with one or more payload agents(e.g., therapeutic agents) is administered to a subject by, or isapplied to, a device, medical device, implant, dental implant, breastimplant, prosthesis, needle, stent, or catheter. Additional methods ofadministration are described in, e.g., Patent Application Nos.US2011-0002880; WO/2008/073395; US2011/0201541; US 2014-0329914; US2015-0105336; WO/2014/136081; WO/2014/141143, and U.S. Pat. No.7,846,891, each of which are herein incorporated by reference in theirentirety for any and all purposes.

Additional methods and embodiments can be found in WO 2017/120092, whichis herein incorporated by reference in its entirety for any and allpurposes.

6. Air Leakage Occlusion

In some embodiments, the self-assembling peptides and pharmaceuticalcompositions described herein may be used for air leakage occlusion. Insome embodiments, the self-assembling peptides and pharmaceuticalcompositions described herein may be used for treating a pulmonarybulla. In embodiments, a method of treating a pulmonary bulla and/or anair leakage in a subject is provided. The method may compriseintroducing a delivery device to a target area of the pulmonary bulla ofthe subject. The method may also comprise positioning an end of thedelivery device in the target area in which treatment of a pulmonarybulla is desired. The method may also comprise administering through thedelivery device a solution comprising a self-assembling peptide orpharmaceutical composition described here in an effective amount and inan effective concentration to the target area to form a barrier underphysiological conditions of the target area to treat the pulmonarybulla. The method may also comprise removing the delivery device fromthe target area.

Additional methods and embodiments can be found in WO 2013/030673, WO2015/138473, U.S. Pat. No. 10,245,299, each of which are hereinincorporated by reference in their entirety for any and all purposes.

7. Prevention and Reduction of Adhesion

In some embodiments, the self-assembling peptides and pharmaceuticalcompositions described herein may be used for preventing or mitigatingbiological tissue adhesion. In various embodiments, the inventionprovides a method for mitigating adhesion to a biological tissue, themethod comprising administering an effective amount of theself-assembling peptides and pharmaceutical compositions describedherein to the biological tissue, wherein the self-assembling peptidesand pharmaceutical compositions described herein mitigates adhesion tothe biological tissue.

In various aspects, the invention provides a method of promotinganti-adhesion, comprising: introducing a delivery device to a targetarea; positioning an end of the delivery device in the target area atwhich anti-adhesion is desired; administering through the deliverydevice a solution comprising a self-assembling peptide or pharmaceuticalcomposition described herein in an effective amount and in an effectiveconcentration to the target area to promote anti-adhesion; and removingthe delivery device from the target area.

In some embodiments the biological tissue comprises an epicardium,intraperitoneum, cecum, intestine, preferentially large intestine,and/or colon. In some embodiments, the target area comprises anepicardium, intraperitoneum, cecum, intestine, preferentially largeintestine, and/or colon.

Additional methods and embodiments can be found in US 2019-0091376 A1,which is herein incorporated by reference in its entirety for any andall purposes.

8. In Vitro Cell Culture

In some embodiments, the self-assembling peptides and pharmaceuticalcompositions described herein may be used for 2D cell culture and/or 3Dcell culture.

9. Bone Void Filler

In some embodiments, the self-assembling peptides and pharmaceuticalcompositions described herein may be used for filling a bone void.

In some embodiments, a method of filling a bone void in a subject isprovided. In some embodiments, the method may involve introducing adelivery device to a bone of a subject, positioning an end of thedelivery device proximate a void in the bone where promotion of bonegrowth is desired, administering through the delivery device a solutioncomprising a self-assembling peptide or pharmaceutical composition ofthe disclosure in an effective amount and in a concentration in a rangeof about 0.1 w/v percent to about 5 w/v percent peptide to form ahydrogel scaffold under physiological conditions to promote bone growthat the target site, and removing the delivery device from the subject.

In one or more embodiments, the self-assembling peptides andpharmaceutical compositions described herein may be used as a bone voidfiller (BVF) that resorbs and is replaced with bone during a healingprocess following administration at a target site. The peptide hydrogelmay be placed into bony voids or gaps of the skeletal system. In certainembodiments, self-assembling peptides and self-assembled structuresthereof may be used as cell culture supports for the repair andreplacement of various tissues and as a scaffold to encapsulate livingcells. The self-assembling peptides and pharmaceutical compositionsdescribed herein may promote tissue regeneration and the production ofrelated extracellular matrix proteins. In at least some embodiments, theself-assembling peptides and pharmaceutical compositions describedherein are non-immunogenic and represents an improvement over existingmaterials for this indication, including demineralized freeze-dried boneallograft (DFDBA) preparations.

Additional methods and embodiments can be found in US 2017-0128622 A1,which is herein incorporated by reference in its entirety for any andall purposes.

10. Artificial Tears for Dry Eye

In some embodiments, the self-assembling peptides and pharmaceuticalcompositions described herein may be used to treat dry eye. In someembodiments, the self-assembling peptides and pharmaceuticalcompositions described herein may be used for artificial tears.

11. Articular Cartilage Repair

In some embodiments, the self-assembling peptides and pharmaceuticalcompositions described herein may be used to repair articular cartilage.In some embodiments, the self-assembling peptides and pharmaceuticalcompositions described herein may be used as an injectable composition.Without being bound by theory, injectable hydrogels comprising theself-assembling peptides and pharmaceutical compositions describedherein can enhance its therapeutic efficacy and improve its ease ofadministration. An ideal injectable scaffold for cartilage regenerationshould typically meet the following criteria: (i) ease of administrationunder physiological conditions, (ii) guaranteed injectability (gelationupon injection via either chemical or physical cross-linking), (iii)excellent biocompatibility and potential biodegradability, (iv) theability to mimic cartilaginous ECM features and promote chondrogenicpotential of cells, (v) the ability to easily fill defect sites insidethe joint and integrate with the surrounding native cartilage tissuerather than shifting readily and (vi) a sustained release profile ifassociated with local drug delivery.

12. Cosmetic Use (a Dermal Filler in Cosmetic Surgery)

In some embodiments, the self-assembling peptides and pharmaceuticalcompositions described herein may be used as a dermal filler. In someembodiments, the self-assembling peptides and pharmaceuticalcompositions described herein may be used as an injectable composition.Without being bound by theory, injectable hydrogels comprising theself-assembling peptides and pharmaceutical compositions describedherein can enhance its therapeutic efficacy and improve its ease ofadministration. An ideal injectable scaffold for cartilage regenerationshould typically meet the following criteria: (i) ease of administrationunder physiological conditions, (ii) guaranteed injectability (gelationupon injection via either chemical or physical cross-linking), (iii)excellent biocompatibility and potential biodegradability, (iv) theability to mimic cartilaginous ECM features and promote chondrogenicpotential of cells, (v) the ability to easily fill defect sites insidethe joint and integrate with the surrounding native cartilage tissuerather than shifting readily and (vi) a sustained release profile ifassociated with local drug delivery.

In some embodiments, the self-assembling peptides and pharmaceuticalcompositions described herein may be used to plump lips. In someembodiments, the self-assembling peptides and pharmaceuticalcompositions described herein may be used to fill or smooth wrinkles. Insome embodiments, the self-assembling peptides and pharmaceuticalcompositions described herein may be used soften facial creases. In someembodiments, the self-assembling peptides and pharmaceuticalcompositions described herein may be used to enhance shallow contours.In some embodiments, the self-assembling peptides and pharmaceuticalcompositions described herein may be used to reconstruct contourdeformities in the face of a subject. In some embodiments, theself-assembling peptides and pharmaceutical compositions describedherein may be used to improve the appearance of recessed scars. In someembodiments, the self-assembling peptides and pharmaceuticalcompositions described herein may be used to decrease or remove theshadow beneath the lower eye lids.

Kits

In another aspect, the disclosure provides kits and articles ofmanufacture comprising the pharmaceutical compositions and/or theself-assembling peptides described herein. The kits may includedirections for the appropriate preparation and/or use of the componentstherein. The kits may further include useful tools for preparationand/or delivery of the pharmaceutical compositions or self-assemblingpeptides to subject (e.g., a human subject), as described herein.

In some embodiments, the kit contains separate containers, dividers orcompartments for the pharmaceutical composition and informationalmaterial. For example, the pharmaceutical composition can be containedin a bottle, vial, or syringe, and the informational material can becontained in a plastic sleeve or packet. In some embodiments, theseparate elements of the kit are contained within a single, undividedcontainer. For example, the pharmaceutical composition orself-assembling peptide may be contained in a bottle, vial or syringethat has attached thereto the informational material in the form of alabel.

In some embodiments, the kit comprises a plurality, e.g., a pack, ofindividual containers, each containing one or more unit dosage forms ofthe pharmaceutical composition or self-assembling peptide. For example,the kit may comprise a plurality of syringes, ampules, foil packets, orblister packs, each containing a single unit dose of the pharmaceuticalcomposition or self-assembling peptide. In some embodiments, a componentof the kit is stored in a sealed vial, for example, with a rubber orsilicone closure (for example, a polybutadiene or polyisoprene closure).In some embodiments, a component of the kit is stored under inertconditions (for example, under nitrogen or another inert gas such asargon). In some embodiments, a component of the kit is stored underanhydrous conditions (for example, with a desiccant). In someembodiments, a component of the kit is stored in a light blockingcontainer such as an amber vial. In some embodiments, the containers ofthe kits can be air tight and/or waterproof.

The kits may further comprise solutions including tonicity agents. Thesesolutions may be packaged separately, or in combination with,pharmaceutical compositions comprising a self-assembling peptideprovided herein.

Articles of manufacture, including for example, syringes,auto-injectors, tubing, and catheters (e.g., with or without aguidewire) are also provided. In some embodiments, the articles ofmanufacture may be pre-filled with a self-assembling peptide, orpharmaceutical composition described herein. These articles ofmanufacture may be separate from or included in a kit described herein.

Further aspects and embodiments of the present invention are set out inthe following numbered paragraphs:

1. A pharmaceutical composition comprising a self-assembling peptide,wherein the self-assembling peptide comprises an amino acid sequence asset forth in:

[(X)i(Y)j(Z)k(Y)l]m(X)n  Formula I,

[(Y)i(X)j(Y)k(Z)l]m(Y)n  Formula II,

[(Z)i(Y)j(X)k(Y)l]m(Z)n  Formula III, or

[(Y)i(Z)j(Y)k(X)l]m(Y)n  Formula IV,

wherein each (X) is independently an ionic, polar amino acid, whereineach (Y) is independently a hydrophobic amino acid, wherein each (Z) isindependently a non-ionic, polar amino acid, wherein each i, j, k, and lis independently an integer ≥1, wherein m is an integer ≥2, and whereinn=0 or an integer ≥1.

2. The pharmaceutical composition of paragraph 1, wherein theself-assembling peptide comprises an amino acid sequence as set forth inFormula I.

3. The pharmaceutical composition of paragraph 1, wherein theself-assembling peptide comprises an amino acid sequence as set forth inFormula II.

4. The pharmaceutical composition of paragraph 1, wherein theself-assembling peptide comprises an amino acid sequence as set forth inFormula III.

5. The pharmaceutical composition of paragraph 1, wherein theself-assembling peptide comprises an amino acid sequence as set forth inFormula IV.

6. The pharmaceutical composition of any one of paragraphs 1-5, whereineach (X) is a basic amino acid.

7. The pharmaceutical composition of paragraph 6, wherein the basicamino acid is selected from the group consisting of arginine, lysine,histidine and ornithine.

8. The peptide of any one of paragraphs 1-5, wherein each (X) is anacidic amino acid.

9. The peptide of paragraph 8, wherein the acidic amino acid is selectedfrom the group consisting of aspartic acid and glutamic acid.

10. The pharmaceutical composition of any one of paragraphs 1-9, whereineach (Y) is selected from the group consisting of alanine, valine,leucine, isoleucine, methionine, phenylalanine, tryptophan, and glycine.

11. The pharmaceutical composition of any one of paragraphs 1-10,wherein each (Z) is selected from the group consisting of the groupconsisting of serine, threonine, tyrosine, cysteine, glutamine,asparagine, and methionine.

12. The pharmaceutical composition of any one paragraphs 1-11, whereinat least one of i, j, k, and l is independently an integer of 1.

13. The pharmaceutical composition of any one of paragraphs 1-12,wherein at least one of i, j, k, and l is independently an integer of 2.

14. The pharmaceutical composition of any one of paragraphs 1-13,wherein m is independently an integer of 2 or 3.

15. The pharmaceutical composition of any one of paragraphs 1-14,wherein the self-assembling peptide comprises an amino acid sequence asset forth in SEQ ID NOs: 1-20.

16. The pharmaceutical composition of any one of paragraphs 1-15,wherein the self-assembling peptide comprises an N-terminal functionalgroup, a C-terminal functional group, or both.

17. The pharmaceutical composition of paragraph 16, wherein theN-terminal functional group is selected from the group consisting of anacetyl, a formyl, pyroglutamyl (pGlu), biotin, polyethylene glycol(PEG), urea, alkylamine, a carbamate, a sulfonamide, dansyl,2,4-dintrophenyl, fluorescein, 7-methoxycoumarin acetic acid,9-fluorenylmethyloxycarbonyl, palmitic acid, succinyl, chloroacetyl,maleimide, benzyloxycarbonyl, bromoacetyl, nitrilotriacetyl,tertbutoxycarbonyl, 4-hydroxyphenylpropionic acid, allyloxycarbonyl,butyric acid, a fatty acid, and trityl.

18. The pharmaceutical composition of paragraph 16, wherein theC-terminal functional group is selected from the group consisting of anamido, an N-alkyl amide, an aldehyde, an ester, an alcohol,para-nitroanilide (pNA), 7-amino-4-methylcoumarin (Amc), a hydrazide,hydroxamic acid, chloromethylketone, p-nitroaniline, para-nitrophenol,hydroxysucinimide ester, fluoromethylketone, cysteamide,9-fluorenemethyl (Fm) ester, allyl ester, 2,4-dimethoxybenzyl ester,2-phenylisopropyl ester, p-nitrobenzyl ester, and 2-chlorotrityl ester.

19. The pharmaceutical composition of any one of paragraphs 1-16,wherein the self-assembling peptide comprises an amino acid sequence asset forth in SEQ ID NOs: 21-40.

20. The pharmaceutical composition of any one of paragraphs 1-19,wherein the self-assembling peptide further comprises at least onebiologically-active peptide motif.

21. The pharmaceutical composition of any one of paragraphs 1-20,wherein the at least one biologically-active peptide motif is present atthe N-terminal end of the self-assembling peptide.

22. The pharmaceutical composition of any one of paragraphs 1-20,wherein the at least one biologically-active peptide motif is present atthe C-terminal end of the self-assembling peptide.

23. The pharmaceutical composition of any one of paragraphs 20-22,wherein the at least one biologically-active peptide motif is derivedfrom laminin-1, collagen IV, fibronectin, elastin, bone marrow homingpeptide 1, bone marrow homing peptide 2, or myelopeptide.

24. The pharmaceutical composition of any one of paragraphs 20-22,wherein the at least one biologically-active peptide motif comprises anamino acid sequence as set forth in any one of SEQ ID NOs: 41-70.

25. The pharmaceutical composition of any one of paragraphs 1-20,wherein the self-assembling peptide comprises an amino acid sequence asset forth in SEQ ID NOs: 71-90.

26. The pharmaceutical composition of any one of paragraphs 1-25,further comprising a tonicity agent.

27. The pharmaceutical composition of paragraph 26, wherein the tonicityagent is present at a concentration of about 0.01 M to about 0.3 M.

28. The pharmaceutical composition of paragraph 26, wherein the tonicityagent is present at a concentration of about 0.15 M.

29. The pharmaceutical composition of any one of paragraphs 1-28, havinga pH of from about 6 to about 8.

30. The pharmaceutical composition of any one of paragraphs 1-28, havinga pH of from about 7 to about 7.5.

31. The pharmaceutical composition of paragraph 29 or paragraph 30,wherein the net charge of the self-assembling peptide is greater than orequal to +1 or less than or equal to −1.

32. The pharmaceutical composition of paragraph 29 or paragraph 30,wherein the net charge of the self-assembling peptide is from about +1to about +6.

33. The pharmaceutical composition of paragraph 29 or paragraph 30,wherein the net charge of the self-assembling peptide is from about −1to about −6.

34. The pharmaceutical composition of any one of paragraphs 1-33,wherein the concentration of the self-assembling peptide from about0.01% (w/v) to about 10% (w/v).

35. The pharmaceutical composition of any one of paragraphs 1-33,wherein the concentration of the self-assembling peptide is from about0.1% (w/v) to about 5% (w/v).

36. The pharmaceutical composition of any one of paragraphs 1-33,wherein the concentration of the self-assembling peptide is from about0.5% (w/v) to about 1.5% (w/v).

37. The pharmaceutical composition of any one of paragraphs 1-33,wherein the concentration of the self-assembling peptide is about 1%(w/v).

38. The pharmaceutical composition of any one of paragraphs 1-37,further comprising an isolated cell.

39. The pharmaceutical composition of paragraph 38, wherein the isolatedcell is a mammalian cell.

40. The pharmaceutical composition of paragraph 38 or paragraph 39,wherein the mammalian cell is an immune cell, a stem cell, chondrocyteprogenitor cells, pancreatic progenitor cells, myoblasts, fibroblasts,keratinocytes, neuronal cells, glial cells, astrocytes, pre-adipocytes,adipocytes, vascular endothelial cells, endothelial progenitor cells,mesenchymal cells, neural stem cells, immune cells, (e.g., B-cells andT-cells), smooth muscle progenitor cells, cardiac myocytes, fetal dermalfibroblasts, epidermal keratinocytes, myoblasts, and capillaryendothelial cells.

41. The pharmaceutical composition of any one of paragraphs 1-40,wherein the pharmaceutical composition further comprises a bioactiveagent.

42. The pharmaceutical composition of paragraph 41, wherein thebioactive agent is selected from the group consisting of a hormone, agrowth factor, insulin, an enzyme, an siRNA, an shRNA, an antisense-RNA,an antibiotic, an antibody, and an anti-inflammatory agent.

43. The pharmaceutical composition of any one of paragraphs 1-42,wherein the pharmaceutical composition is an aqueous solution.

44. The pharmaceutical composition of any one of paragraphs 1-42,wherein the pharmaceutical composition is a hydrogel.

45. The pharmaceutical composition of paragraph 44, wherein thepharmaceutical composition is a hydrogel, and wherein the hydrogelcomprises a storage modulus of at least about 10 Pascal (Pa).

46. An article of manufacture comprising the pharmaceutical compositionof any one of paragraphs 1-45.

47. The article of manufacture of paragraph 46, wherein the article is asyringe, a vial, an auto-injector, tubing, or a catheter.

48. A method of treating a subject in need thereof, comprisingadministering an effective amount of the pharmaceutical compositionaccording to any one of paragraphs 1-45 to the subject.

49. A method of promoting tissue repair or regeneration in a subject inneed thereof, comprising contacting a tissue of the subject with thepharmaceutical composition according to any one of paragraphs 1-45,thereby promoting tissue repair or regeneration of the tissue.

50. The method of paragraph 49, wherein the tissue is skin, bone,cartilage, neural tissue, ligament, tendon, vascular tissue, or muscle.

51. The method of paragraph 49, wherein the tissue is optic tissue.

52. The method of paragraph 49, wherein the tissue is cardiac tissue.

53. The method of any one of paragraphs 49-52, wherein the subject has acongenital disease or disorder resulting in a need for the tissue repairor regeneration.

54. The method of any one of paragraphs 49-52, wherein the subject hassuffered an injury resulting in a need for the tissue repair orregeneration.

55. The method of paragraph 54, wherein the injury is a result ofsurgery, trauma, stroke, tumor, or a disease or disorder.

56. A method of promoting wound healing in a subject in need thereof,comprising contacting a wound of the subject with the pharmaceuticalcomposition according to any one of paragraphs 1-45, thereby promotingwound healing.

57. The method of paragraph 56, wherein the wound comprises an abrasion,a burn, a chap, a detrition, a cut, an ulcer, a laceration, an incision,or a scratch.

58. A method of stopping or preventing a bleeding at a site within asubject, comprising contacting the site with the pharmaceuticalcomposition according to any one of paragraphs 1-45, wherein thepharmaceutical composition creates a physical barrier thereby stoppingor preventing bleeding at the site within the subject.

59. A method of excising a lesion from a site in the gastrointestinaltract of a subject, comprising:

a. contacting submucosa below the lesion with a pharmaceuticalcomposition according to any one of paragraphs 1-45, thereby lifting thelesion; and

b. excising the lesion from the site in the gastrointestinal tract ofthe subject.

60. The method of paragraph 59, wherein the lesion comprises a polyp, anulcer, or a tumor.

61. The method of paragraph 59 or paragraph 60, wherein the lesion ispresent in a region of the gastrointestinal tract of the subjectselected from a mouth, a throat, an esophagus, a stomach, a smallintestine, a large intestine, a colon, and a rectum.

62. A method of culturing a cell, comprising contacting the cell withthe pharmaceutical composition according to any one of paragraphs 1-45.

The following examples are illustrative and not restrictive. Manyvariations of the technology will become apparent to those of skill inthe art upon review of this disclosure. The scope of the technologyshould, therefore, be determined not with reference to the examples, butinstead should be determined with reference to the appended claims alongwith their full scope of equivalents.

EXAMPLES Materials and Methods

The following materials and methods were utilized to conduct theexperiments described in the Examples below.

Peptide Synthesis

All peptides utilized in the Examples herein were synthesized byconventional solid peptide synthesis using an automated peptidesynthesizer.

Viscosity Determination

Peptides formulated as aqueous solutions (200 μL), were placed betweenthe plates of a rheometer (DHR-1, TA Instruments, 20 mm plates at ameasuring geometry of 500 μm) and the viscosity was measured from 0.001s⁻¹ to 100 s⁻¹ shear rate. Thixotropic Measurements

Peptides formulated as aqueous solutions (200 μL), were placed betweenthe plates of a rheometer (DHR-1, TA Instruments, 20 mm plates at ameasuring geometry of 500 μm) and the sample was exposed to a fast platerotation at 1000 s⁻¹ of shear rate for 1 minute and the storage moduluswas measured at 1 rad s⁻¹ angular frequency for 10 minutes.

Frequency Sweep Test

Peptides formulated as aqueous solutions were placed between the platesof a rheometer (DHR-1, TA Instruments, 20 mm plates at a measuringgeometry of 500 μm or 40 mm cone and plate at a measuring geometry of2.0° of cone angle) and frequency stress sweep tests were performed at0.1 Hz to 10 Hz of frequency with 0.1% of strain; measurements wereperformed after 2 minutes of relaxation time at 37° C. To do frequencysweep tests after exposure to Dulbecco's Modified Eagle Medium (DMEM),10 mL of DMEM was added in chamber surrounding the plates, the sample inplates was submerged into the DMEM and the frequency test was performedas described after 20 minutes.

Endoscopic Mucosal Resection and Endoscopic Submucosal Dissection

An in vivo porcine animal model was used to perform endoscopic mucosaland submucosal dissections as described in Uraoka et al. (2009) DrugDes. Devel. Ther. 2:131-8, incorporated herein by reference. Briefly, 2mL of select peptide formulations were injected in between the muscleand submucosal layer in stomach of a pig and the submucosa was dissectedusing electro-knife. The gross appearance of the injection site and ofdissected submucosal tissues at the injection sites were analyzed.

Example 1. Determination of Net Charge of Exemplary Self-AssemblingPeptides in Formulations at Varying pH Ranges

Several exemplary self-assembling peptides described herein (Table 5;SEQ ID NO:21-40) were designed to have a net charge, positive ornegative, when formulated at pH 7.5. In contrast, previously-describedself-assembling peptides have a net zero charge at the same pH. The netcharges of the exemplary self-assembling peptides in solution at pH 7.5was determined and is shown in Table 5. While the previously describedself-assembling peptides RADA16, IEIK13, and KLD12 (SEQ ID NO:91-93,respectively) have about zero net charge at pH 7.5, the newly developedself-assembling peptides had a net positive or net negative charge atthe same pH.

As shown in FIGS. 1-3 , this net charge behavior extends around the testpoint of pH 7.5 shown in Table 5. For example, FIG. 1 shows net chargeas a function of pH for KLNL12 (SEQ ID NO:21, about +3 around neutralpH). FIG. 2 shows net charge as a function of pH for NLEL12 (SEQ IDNO:33, about −3 around neutral pH). FIG. 3 shows net charge as afunction of pH for RADA16 (SEQ ID NO:91, about 0 around neutral pH).

TABLE 5 Net charge of exemplary self-assembling peptides at pH 7.5. SEQNet charge Name ID NO: Sequence at pH 7.5 KLNL12 21 Ac-KLNLKLNLKLNL-NH2+3 LNLK12 22 Ac-LNLKLNLKLNLK-NH2 +3 NLKL12 23 Ac-NLKLNLKLNLKL-NH2 +3LKLN12 24 Ac-LKLNLKLNLKLN-NH2 +3 KLNL13 25 Ac-KLNLKLNLKLNLK-NH2 +4KLNL17 26 Ac-KLNLKLNLKLNLKLNLK-NH2 +5 IQIK12 27 Ac-IQIKIQIKIQIK-NH2 +3IQIK13 28 Ac-IQIKIQIKIQIKI-NH2 +3 KIQI13 29 Ac-KIQIKIQIKIQIK-NH2 +4QIKI13 30 Ac-QIKIQIKIQIKIQ-NH2 +3 IKIQ13 31 Ac-IKIQIKIQIKIQI-NH2 +3INIK13 32 Ac-INIKINIKINIKI-NH2 +3 NLEL12 33 Ac-NLELNLELNLEL-NH2 −3NLDL12 34 Ac-NLDLNLDLNLDL-NH2 −3 KANA12 37 Ac-KANAKANAKANA-NH2 +3 KVNV1238 Ac-KVNVKVNVKVNV-NH2 +3 RANA16 39 Ac-RANARANARANARANA-NH2 +4 KLTL12 40Ac-KLTLKLTLKLTL-NH2 +3 RADA16* 91 Ac-RADARADARADARADA-NH2  0 IEIK13* 92Ac-IEIKIEIKIEIKI-NH2  0 KLDL12* 93 Ac-KLDLKLDLKLDL-NH2  0 *exemplaryconventional self-assembling peptides for comparison

FIGS. 4-5 provide molecular models of the atomic structure and netcharge of exemplary self-assembling peptides. FIG. 4 shows that RANA16(SEQ ID NO:39) has a net charge of +4 at pH 7.5. In contrast, FIG. 5shows that RADA16 (SEQ ID NO:91) has a net charge of 0 at pH 7.5. Thestructures were computationally simulated using MarvinSketch and bothexhibit β-sheet conformations. Despite RANA16 (SEQ ID NO:39) and RADA 16(SEQ ID NO:91) exhibiting similar structural conformations at pH 7.5,the net charge of the two peptides is markedly different, with RANA16(SEQ ID NO:39) having a +4 net positive charge, while RADA16 (SEQ IDNO:91) has zero net charge.

Example 2. Characterization of Solubility of Exemplary Self-AssemblingPeptides in Aqueous Pharmaceutical Compositions Formulated at pH 2.5 and7.5

Several exemplary self-assembling peptides described herein (see Table5; SEQ ID NO:21-40) were designed to: have a non-zero net charge, havehigh solubility when formulated in solution at pH 7.5, and transitionfrom liquid state to hydrogel after administration (e.g., afterinjection into a subject or admixture with solutions containing specificconcentrations of tonicity agents (e.g., salts)). In contrast,previously described self-assembling peptides RADA16, IEIK13, and KLDL12(SEQ ID NO:91-93, respectively) have a zero net charge and must beformulated in solution at acidic pH in order to retain their ability toconditionally transition from liquid state to hydrogel afteradministration.

To determine the solubility of exemplary self-assembling peptides inaqueous pharmaceutical compositions formulated at acidic and neutral pH,the appearance of the pharmaceutical compositions was assessed.Homogenous and transparent appearance reflects sufficient solubility ofthe peptides in solution and minimal hydrogel formation. Phaseseparation and cloudy appearance reflects poor solubility of thepeptides in solution and/or the formation of hydrogel.

The appearance of exemplary self-assembling peptides at pH 2.5 and pH7.5 is shown below in Table 6 and FIG. 20 . Aqueous pharmaceuticalcompositions at pH 7.5 comprising the newly described self-assemblingpeptides were transparent and homogenous, while solutions of previouslydisclosed self-assembling peptides RADA16 (SEQ ID NO:91), IEIK13 (SEQ IDNO:92) and KLDL12 (SEQ ID NO:93) at pH 7.5 exhibited phase separationand cloudy appearance (see Table 6 and FIGS. 20A-C).

TABLE 6 Appearance of exemplaryself-assembling peptides at pH 2.5 and pH 7.5. SEQ IDAppearance at different pH NO: Name Sequence At pH 2.5 At pH 7.5 21KLNL12 Ac-KLNLKLNLKLNL-NH2 Homogeneous Homogeneous and transparentand transparent 22 LNLK12 Ac-LNLKLNLKLNLK-NH2 Homogeneous Homogeneousand transparent and transparent 23 NLKL12 Ac-NLKLNLKLNLKL-NH2Homogeneous Homogeneous and transparent and transparent 24 LKLN12Ac-LKLNLKLNLKLN-NH2 Homogeneous Homogeneous and transparentand transparent 25 KLNL13 Ac-KLNLKLNLKLNLK-NH2 Homogeneous Homogeneousand transparent and transparent 26 KLNL17 Ac-KLNLKLNLKLNLKLNLK-Homogeneous Homogeneous NH2 and transparent and transparent 27 IQIK12Ac-IQIKIQIKIQIK-NH2 Homogeneous Homogeneous and transparentand transparent 28 IQIK13 Ac-IQIKIQIKIQIKI-NH2 Homogeneous Homogeneousand transparent and transparent 29 KIQI13 Ac-KIQIKIQIKIQIK-NH2Homogeneous Homogeneous and transparent and transparent 32 INIK13Ac-INIKINIKINIKI-NH2 Homogeneous Homogeneous and transparentand transparent 33 NLEL12 Ac-NLELNLELNLEL-NH2 Phase-separatedHomogeneous and cloudy and transparent 34 NLDL12 Ac-NLDLNLDLNLDL-NH2Phase-separated Homogeneous and cloudy and transparent 35 QLEL12Ac-QLELQLELQLEL-NH2 Phase-separated Homogeneous and cloudyand transparent 36 LELQ12 Ac-LELQLELQLELQ-NH2 Phase-separatedHomogeneous and cloudy and transparent 91 RADA16 Ac-RADARADARADARADA-Homogeneous Phase-separated NH2 and transparent and cloudy 92 IEIK13Ac-IEIKIEIKIEIKI-NH2 Homogeneous Phase-separated and transparentand cloudy 93 KLDL12 Ac-KLDLKLDLKLDL-NH2 Homogeneous Phase-separatedand transparent and cloudy

Example 3. Characterization of Solubility of Aqueous PharmaceuticalCompositions Formulated at pH 7.5 and Varying Ionic Strengths

The self-assembling peptides described herein (Table 5; SEQ IDNOs:21-40) were designed to: have a non-zero net charge, have highsolubility when formulated as aqueous solutions at pH 7.5 havingisotonic ionic strength (e.g., 0.15 M salt ions), and to transition fromliquid state to hydrogel after administration. To determine thesolubility of aqueous pharmaceutical compositions formulated at pH 7.5in the presence of varying ionic strengths, aqueous pharmaceuticalcompositions having different concentrations of sodium chloride wereprepared, and their appearance assessed. Homogenous and transparentappearance reflects sufficient solubility of the peptides in solutionand minimal hydrogel formation. Phase separation and cloudy appearancereflects poor solubility of the peptides in solution and/or theformation of hydrogel.

As shown in Table 7, in general, the increased amounts of hydrophobicresidues correlated with decreased solubility at higher ionic strengths.Therefore, the amino acid composition of the self-assembling peptidescan be manipulated to achieve desired solubility of at a desired ionicstrength.

TABLE 7 Appearance of exemplaryself-assembling peptides at different ionic strength.Appearance at different ionic strength SEQ(e.g., NaCl concentration) at around pH 7.5 ID 0.15 M NO: Name Sequence0 M 0.05 M 0.1 M (isotonic) 21 KLNL12 Ac- Homogeneous  Homogeneous Homogeneous  Homogeneous  KLNLKLNLKLNL- and and and and NH2 transparenttransparent transparent transparent 22 LNLK12 Ac- Homogeneous Homogeneous  Phase- Phase- LNLKLNLKLNLK- and and separated separated NH2transparent transparent and cloudy and cloudy 23 NLKL12 Ac- Homogeneous Homogeneous  Homogeneous  Homogeneous  NLKLNLKLNLKL- and and and and NH2transparent transparent transparent transparent 24 LKLN12 Ac-Homogeneous  Homogeneous  Phase- Phase- LKLNLKLNLKLN- and and separatedseparated NH2 transparent transparent and cloudy and cloudy 25 KLNL13Ac- Homogeneous  Homogeneous  Homogeneous  Homogeneous  KLNLKLNLKLNLK-and and and and NH2 transparent transparent transparent transparent 26KLNL17 Ac- Homogeneous  Homogeneous  Homogeneous  Homogeneous KLNLKLNLKLNLKL and and and and NLK-NH2 transparent transparenttransparent transparent 27 IQIK12 Ac- Homogeneous  Homogeneous Homogeneous  Homogeneous  IQIKIQIKIQIK- and and and and NH2 transparenttransparent transparent transparent 28 IQIK13 Ac- Homogeneous  Phase-Phase- Phase- IQIKIQIKIQIKI- and separated separated separated NH2transparent and cloudy and cloudy and cloudy 29 KIQI13 Ac- Homogeneous Homogeneous  Homogeneous  Homogeneous  KIQIKIQIKIQIK- and and and andNH2 transparent transparent transparent transparent 32 INIK13 Ac-Homogeneous  Homogeneous  Phase- Phase- INIKINIKINIKI- and and separatedseparated NH2 transparent transparent and cloudy and cloudy 33 NLEL12Ac- Homogeneous  Homogeneous  Homogeneous  Homogeneous  NLELNLELNLEL-and and and and NH2 transparent transparent transparent transparent 34NLDL12 Ac- Homogeneous  Homogeneous  Homogeneous  Homogeneous NLDLNLDLNLDL- and and and and NH2 transparent transparent transparenttransparent 35 QLEL12 Ac- Homogeneous  Homogeneous  Homogeneous Homogeneous  QLELQLELQLEL- and and and and NH2 transparent transparenttransparent transparent 36 LELQ12 Ac- Homogeneous  Homogeneous Homogeneous  Homogeneous  LELQLELQLELQ- and and and and NH2 transparenttransparent transparent transparent 91 RADA16* Ac- Homogeneous Homogeneous  Homogeneous  Homogeneous  RADARADARADARA and and and andDA-NH2 transparent transparent transparent transparent 93 KLDL12* Ac-Homogeneous  Homogeneous  Homogeneous  Homogeneous  KLDLKLDLKLDL- andand and and NH2 transparent transparent transparent transparent 92IEIK13* Ac- Homogeneous  Phase- Phase- Phase- IEIKIEIKIEIKI- andseparated separated separated NH2 transparent and cloudy and cloudyand cloudy *peptides precipitate at around pH 7.5, and were thereforeformulated at around pH 2.2~2.3

Example 4. Determination of Shear Thinning and Thixotropic Behavior ofAqueous Pharmaceutical Compositions Comprising Exemplary Self-AssemblingPeptides Formulated at pH 7.5 at Varying Ionic Strengths

The self-assembling peptides disclosed herein (Table 5 SEQ ID NOs:21-40)were designed to have a non-zero net charge, high solubility whenformulated in aqueous solution at pH 7.5 in presence of isotonic ionicstrength (defined at 0.15M salt ions), and to transition from liquidstate to hydrogel after administration to a subject. In some clinicalapplications, administration of pharmaceutical compositions comprisingself-assembling peptides requires exposing the compositions to pressureand/or shear thinning (e.g., during injection through a syringe needleor transferred via a pump-based system). Ideally, pharmaceuticalcompositions comprising the self-assembling peptides described hereinexhibit reduced viscosity under pressure or shear thinning forces andreturn to basal viscosity after removal from these forces. To determinethe shear thinning properties of pharmaceutical compositions of theself-assembling peptides formulated at pH 7.5, formulations comprisingvarying ionic strengths were prepared using different concentrations ofsodium chloride, and viscosity measured at increasing shear rates.Thixotropic properties of formulations were also determined as well asthe storage modulus over time at specific frequencies and pressure.

FIGS. 6-8 show the experimentally determined viscosity of aqueouspharmaceutical compositions comprising either 1% (w/v) KLNL12 (SEQ IDNO:21), 1% (w/v) IQIK13 (SEQ ID NO:28), or 1% (w/v) NLEL12 (SEQ IDNO:33), at pH 7.5. FIGS. 9 and 10 show the experimentally determinedviscosity of aqueous pharmaceutical compositions comprising either 1%KLNL12 (SEQ ID NO:21) or 1% NLEL12 (SEQ ID NO:33), and 0.9% NaCl (tomimic physiological conditions) at pH 7.5. The data demonstrates thatthe aqueous pharmaceutical compositions exhibit sheer thinning. That is,decreased viscosity with increasing sheer rate. This property mayadvantageously facilitate application and use of the pharmaceuticalcompositions (e.g., due to decreased viscosity while pumping) whilemaintaining a desirable viscosity at a site of application (e.g., whereno sheer force is being applied).

FIGS. 11 and 12 show the thixotropic properties of pharmaceuticalcompositions comprising 1% KLNL12 (SEQ ID NO:21) or 1% NLEL12 (SEQ IDNO:33), and 0.9% NaCl, at pH 7.5, after shear stress is removed. Theaqueous pharmaceutical compositions were flowed at 1000 l/s of shearrate for 1 min and their storage modulus at 10 rad/s of frequency and at0.1 Pa of stress was tested over time. The tested aqueous pharmaceuticalcompositions exhibited thixotropic properties. This property mayadvantageously facilitate application and use of the pharmaceuticalcompositions since they exhibit predictable and reliable propertiesafter the application of mechanical stress.

Example 5. Determination of Rheological Properties of PharmaceuticalCompositions Comprising Self-Assembling Peptides

Rheological properties of aqueous pharmaceutical compositions comprisingthe exemplary self-assembling peptides KLNL12 (SEQ ID NO:21), KIQI13(SEQ ID NO:29), or NLEL12 (SEQ ID NO:33) were determined by performing afrequency sweep test over storage modulus.

FIGS. 13-15 show the rheological properties of aqueous pharmaceuticalcompositions comprising 1% (w/v) of KLNL12 (SEQ ID NO:21) (FIG. 13 ),KIQI13 (SEQ ID NO:29) (FIG. 14 ), or NLEL12 (SEQ ID NO:33) (FIG. 15 ) atpH 7.5, before and after exposure to buffered Dulbecco's modifiedEagle's medium (DMEM) to simulate body fluid. In each case, rheologicalproperties (e.g., mechanical strength) of the peptide solutionsincreased after DMEM treatment.

FIGS. 16-18 show the rheological properties of aqueous pharmaceuticalcompositions comprising 1% (w/v) of KLNL12 (SEQ ID NO:21) (FIG. 16 ),KIQI13 (SEQ ID NO:29) (FIG. 17 ), or NLEL12 (SEQ ID NO:33) (FIG. 18 )having isotonic salinity (0.9% NaCl) at pH 7.5, before and afterexposure to DMEM. The rheological properties (e.g., mechanical strength)of the peptide solutions increased after DMEM treatment.

The data above demonstrates that aqueous pharmaceutical compositionscomprising KLNL12 (SEQ ID NO:21), KIQI13 (SEQ ID NO:29), or NLEL12 (SEQID NO:33) exhibited increased mechanical strength in the presence of aphysiological fluid, and therefore are suitable for therapeuticapplications in vivo.

Example 6. Pharmaceutical Compositions Comprising ExemplarySelf-Assembling Peptides Exhibited Reduced Tissue Damage as Compared toPharmaceutical Compositions of RADA16

Given that the self-assembling peptides disclosed herein (Table 5; SEQID NOs:21-40) have a non-zero net charge when formulated at neutral pH(e.g., pH 7.5), it was hypothesized that these compositions exhibitedreduced adverse effects when administered or contacted with mammaliantissue, as compared to compositions comprising previously describedself-assembling peptides (e.g., RADA16) which are formulated at acidicpH. Therefore, an in vivo porcine model system was used to assess theability of these compositions to induce submucosal elevation uponinjection into gastrointestinal submucosa, and the degree of tissuedamage.

In this model, submucosal injections with liquid compositions wereperformed and the morphology of the site of injection analyzed.Submucosal injection with aqueous solutions is used during the excisionof lesions from the gastrointestinal tract in order to facilitateremoval of the lesion by providing a safety cushion during resection(see, e.g., Kim et al. (2013) World J. Gastroenterol. 19(20): 3069-76,incorporated herein by reference). Here, aqueous pharmaceuticalcompositions comprising three exemplary self-assembling peptidesformulated at pH 7.5: KLNL12 (SEQ ID NO:21), NLKL12 (SEQ ID NO:23) orKIQI13 (SEQ ID NO:29), were compared to an aqueous pharmaceuticalcomposition comprising the previously described RADA16 self-assemblingpeptide (formulated at pH 2.5), a 0.4% solution of sodium hyaluronate(MucoUp®, Boston Scientific Japan K.K., Tokyo, Japan), and saline, pH7.5. As shown in FIGS. 21A and 21B, injection with compositionscomprising KLNL12 (SEQ ID NO:21), NLKL12 (SEQ ID NO:23) or KIQI13 (SEQID NO:29) resulted in submucosal elevation. These compositions showedbetter submucosal elevation than saline and MucoUp® over time.

The effect of injection with aqueous pharmaceutical compositionscomprising RADA16 (SEQ ID NO:91), pH 2.5, with and without 0.9% (w/v)NaCl, on the morphology of submucosal tissue was analyzed afterdissection of tissue at the site of injection using an electro-knife. Asshown in FIGS. 22A and 22B, injection with compositions comprisingRADA16 (SEQ ID NO:91), with and without 0.9% NaCl, resulted in tissuedamaged which included mucosal aggregation visualized as whiteprecipitate near the injection site. In contrast, no tissue damage wasobserved when saline was injected (FIG. 22A).

The effect of injection with aqueous pharmaceutical compositionscomprising the exemplary self-assembling peptides KLNL12 (SEQ ID NO:21),NLEL12 (SEQ ID NO:33), QLEL12 (SEQ ID NO:35), or LELQ12 (SEQ ID NO:36),all with 0.9% (w/v) NaCl, pH 7.5, on the morphology of submucosal tissuewas also analyzed after dissection of tissue at the site of injectionusing an electro-knife. As shown in FIGS. 23A-23D, no mucosalaggregation was observed at the injection sites and the morphology ofthe sites was readily visible.

As an additional control, the effect of injection with phosphatebuffered saline (PBS) at either pH 7.4 or pH 2.5 on the morphology ofsubmucosal tissue was analyzed after dissection of tissue at the site ofinjection using an electro-knife. Mucosal aggregation visualized aswhite precipitate near the injection site was solely observed at sitesinjected with acidic PBS (i.e., pH 2.5) (data not shown), confirmingthat the changes in mucosal morphology appear to be attributable to thepH of the composition.

A summary of the appearance of stomach mucosa after injection with eachcomposition used in this example is provided in Table 8 below.

TABLE 8 Appearance of porcine stomach mucosa at injection sitesAppearance of stomach Injected Composition mucosa Water (neutral pH)clear/transparent Saline (0.9% NaCl) (neutral pH) clear/transparent PBS(pH 7.4) clear/transparent PBS (pH 2.5) cloudy/aggregation 0.2% RADA16(SEQ ID NO: 91) (pH 2.5) cloudy/aggregation 0.2% RADA16 (SEQ ID NO: 91)with cloudy/aggregation NaCl 0.9% (pH 2.5) 0.1% (w/v) KLNL12 (SEQ ID NO:21) clear/transparent with NaCl 0.9% (pH 7.5) 0.1% (w/v) KIQI12 (SEQ IDNO: 99) Clear/transparent with NaCl 0.9% (pH 7.5) 0.2% (w/v) NLEL12 (SEQID NO: 33) clear/transparent with NaCl 0.9% (pH 7.5) 0.2% (w/v) LELQ12(SEQ ID NO: 36) clear/transparent with NaCl 0.9% (pH 7.5) 0.2% (w/v)QLEL12 (SEQ ID NO: 35) clear/transparent with NaCl 0.9% (pH 7.5)

The data presented in this example demonstrate that injections intoporcine stomach submucosal layer with aqueous pharmaceuticalcompositions of RADA16 formulations (SEQ ID NO:91), at pH 2.5, result inmucosal aggregation after a period of about 10-15 minutes. In contrast,injection with aqueous pharmaceutical compositions comprising theexemplary self-assembling peptides KLNL12 (SEQ ID NO:21), KIQI12 (SEQ IDNO:99), NLEL12 (SEQ ID NO:33), QLEL12 (SEQ ID NO:35), or LELQ12 (SEQ IDNO:36), all at pH 7.5, do not appear to alter submucosal tissuemorphology. This result is consistent with the notion that acidiccompositions (e.g., those containing RADA16) damage the mucosal tissue,while injection with compositions at neutral pH results in less tissuedamage.

In another embodiment, mucosal aggregation visualized as whiteprecipitate near the injection site was evaluated in an in vivo porcineesophagus.

A summary of the appearance of esophagus mucosa after injection witheach composition used in this example is provided in Table 9 below.

Mucosal aggregation was observed at sites injected with KIQI12 (SEQ IDNO:99) as well as acidic PBS (pH 2) and RADA16 (SEQ ID NO:91). KIQI12(SEQ ID NO:99) is a representative peptide among the positively chargedpeptides at physiological pH. However, mucosal aggregation was notobserved with NLEL12 (SEQ ID NO:33), QLEL12 (SEQ ID NO:35), and LELQ12(SEQ ID NO:36), which are negatively charged at physiological pH. Thisresult demonstrates that the changes in mucosal morphology appear to beattributable to both pH and the positive or negative charge of thecomposition at physiological pH in some submucosal tissues comprisingesophagus.

TABLE 9 Appearance of porcine esophagus mucosa at injection sitesAppearance of stomach Injected Composition mucosa Water (neutral pH)clear/transparent Saline (0.9% NaCl) (neutral pH) clear/transparent PBS(pH 7.4) clear/transparent PBS (pH 2.5) cloudy/aggregation 0.2% RADA16(SEQ ID NO: 91) (pH 2.5) cloudy/aggregation 0.2% RADA16 (SEQ ID NO: 91)with cloudy/aggregation NaCl 0.9% (pH 2.5) 0.1% (w/v) KIQI12 (SEQ ID NO:99) cloudy/aggregation with NaCl 0.9% (pH 7.5) 0.2% (w/v) NLEL12 (SEQ IDNO: 33) clear/transparent with NaCl 0.9% (pH 7.5) 0.2% (w/v) LELQ12 (SEQID NO: 36) clear/transparent with NaCl 0.9% (pH 7.5) 0.2% (w/v) QLEL12(SEQ ID NO: 35) clear/transparent with NaCl 0.9% (pH 7.5)

Example 7, Pharmaceutical Compositions with Increased MechanicalStrength

In some embodiments, peptides formulated as aqueous solutions, volume700 μl, were placed between the plates of a rheometer (DHR-1, TAInstruments, 40 mm cone and plate at a measuring geometry of 2.0° ofcone angle) and frequency sweep tests were performed at 0.1 Hz to 10 Hzof frequency with 0.1% of strain; measurements were performed after 2minutes of relaxation time at 37° C. In some embodiments, frequencysweep tests were performed after exposure to Dulbecco's Modified EagleMedium (DMEM), where 10 mL of DMEM was added in chamber surrounding theplates, the sample in plates was submerged into the DMEM and thefrequency test was performed as described after 20 minutes.

FIG. 24A shows changes in rheological properties of an aqueouspharmaceutical compositions comprising 0.15% (w/v) QLEL12 (SEQ ID NO:35)with 0.9% NaCl (w/v) at pH 7.5, as reflected by increased mechanicalstrength after exposure to DMEM. At a frequency of 0.1 Hz, themechanical strength of the composition comprising a self-assemblingpeptide was increased 23-fold. At a frequency of 1 Hz, the mechanicalstrength of the composition comprising a self-assembling peptide wasincreased 20-fold. At a frequency of 10 Hz, the mechanical strength ofthe composition comprising a self-assembling peptide was increased9-fold.

FIG. 24B shows rheological properties of an aqueous pharmaceuticalcompositions comprising QLEL12 (SEQ ID NO:35) with 0.9% NaCl (w/v) at pH7.5 at various concentrations between 0.1% (w/v) and 0.3% (w/v). Therewas a linear relationship between the rheological property andconcentration of the self-assembling peptide. Increased concertation ofthe self-assembling peptide within the composition resulted in increasedmechanical strength.

Example 8, Pharmaceutical Compositions with Elevated Submucosal Layerand Increased Lifting Capability

In some embodiments, the capability of peptide solution to elevate thesubmucosal layer was evaluated using an ex-vivo canine model. For thistest, QLEL12 (SEQ ID NO:35) was selected because it showed goodrheological properties as well as no unclear/cloudy sites in thesubmucosa layer. QLEL12 (SEQ ID NO:35) solutions of 0.1%, 0.15%, 0.2%,0.3% (w/v) were prepared with 0.9% NaCl at pH 7.5. Normal saline wasused as a reference solution. Each sample of 0.5 mL was injected to thesubmucosal layer of either stomach and colon in the ex-vivo caninemodel. The elevation heights were measured using electronic caliper atthe point of 0 min, 10 min, 20 min, 30 min, 40 min, 50 min and 60 minafter the initial injection. The elevation heights were measured usingelectronic caliper after each sample solution was injected using asyringe needle under the submucosal layer of stomach and colon. Theelevation height is a distance between the bottom of the flat surface ofskin and the top of the swollen bulla after injection under thesubmucosal layer.

The lifting capability of injected solution to lift the submucosal layerfrom the muscle layer after the solution is injected was measured. Insome embodiments, the muscle layer was gastrointestinal. If initialelevation heights and overall elevation heights over time are highenough for submucosal removal procedure, this is considered to have goodlifting capability.

FIG. 25A shows elevation heights of the submucosal layer in the caninestomach after injecting 0.5 mL of QLEL12 (SEQ ID NO:35) with 0.9% NaCl(w/v) at pH 7.5 at various concentrations between 0.1% (w/v) and 0.3%(w/v).

FIG. 25B shows relative elevation value to the initial height in thecanine colon after injecting 0.5 mL of QLEL12 (SEQ ID NO:35) with 0.9%NaCl (w/v) at pH 7.5 at various concentrations between 0.1% (w/v) and0.3% (w/v).

FIG. 25C shows elevation heights of the submucosal layer in the caninecolon after injecting 0.5 mL of QLEL12 (SEQ ID NO:35) with 0.9% NaCl(w/v) at pH 7.5 at various concentrations between 0.1% (w/v) and 0.3%(w/v).

FIG. 25D shows relative elevation value to the initial height in thecanine colon after injecting 0.5 mL of QLEL12 (SEQ ID NO:35) with 0.9%NaCl (w/v) at pH 7.5 at various concentrations between 0.1% (w/v) and0.3% (w/v).

Treatment with a composition comprising a representative self-assemblingpeptide resulted in increased retention of elevation heights of thesubmucosal layer in both the colon and stomach for all concentrationstested. Treatment with a composition comprising a representativeself-assembling peptide also resulted in increased retention of relativeelevation of the submucosal layer in both the colon and stomach for allconcentrations tested. The lifting capability of a compositioncomprising a representative self-assembling peptide also showed goodlifting capability, which aids in ease of removal of certain sections ofthe submucosal layer.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present invention; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

1-45. (canceled)
 46. A method of making a self-assembling peptide,wherein the self-assembling peptide comprises the amino acid sequenceQLEL12 (SEQ ID NO:35), wherein the method comprises synthesizing theself-assembling peptide and providing it in a power or a solution form.47. The method of claim 46, comprising including in the peptide one orboth of, a) an N-terminal functional group selected from the groupconsisting of an acetyl, a formyl, pyroglutamyl (pGlu), biotin,polyethylene glycol (PEG), urea, alkylamine, a carbamate, a sulfonamide,dansyl, 2,4-dintrophenyl, fluorescein, 7-methoxycoumarin acetic acid,9-fluorenylmethyloxycarbonyl, palmitic acid, succinyl, chloroacetyl,maleimide, benzyloxycarbonyl, bromoacetyl, nitrilotriacetyl,tertbutoxycarbonyl, 4-hydroxyphenylpropionic acid, allyloxycarbonyl,butyric acid, a fatty acid, and trityl, and b) a C-terminal functionalgroup selected from the group consisting of an amine, an amido, anN-alkyl amide, an aldehyde, an ester, an alcohol, para-nitroanilide(pNA), 7-amino-4-methylcoumarin (Amc), a hydrazide, hydroxamic acid,chloromethylketone, p-nitroaniline, para-nitrophenol, hydroxysucinimideester, fluoromethylketone, cysteamide, 9-fluorenemethyl (Fm) ester,allyl ester, 2,4-dimethoxybenzyl ester, 2-phenylisopropyl ester,p-nitrobenzyl ester, and 2-chlorotrityl ester.
 48. The method of claim46, comprising including in the peptide at least one biologically activepeptide motif at the N-terminal end, or the C-terminal end, or both, ofthe self-assembling peptide.
 49. The method of claim 48, comprisingincluding in the peptide at least one biologically-active peptide motifis derived from laminin-1, collagen IV, fibronectin, elastin, bonemarrow homing peptide 1, bone marrow homing peptide 2, or myelopeptide.50. A method of making a pharmaceutical composition, comprising adding aself-assembling peptide according to claim
 46. 51. The method of claim50, comprising adding a tonicity agent wherein the tonicity agentcomprises: one or more salts selected from the group consisting of NaCl,KCl, MgCl₂, CaCl₂, NH₄Cl, Na₂HPO₄, KH₂PO₄, and CaSO₄; and/or one or moresugars selected from the group consisting of dextrose, mannitol,glycerin, sucrose, and trehalose.
 52. The method of claim 51, whereinthe tonicity agent comprises one or more salts and is present in aconcentration of about 0.01 M to about 0.3 M or about 0.15 M.
 53. Themethod of claim 51, wherein the tonicity agent comprises one or moresugars and is present in a concentration of about 0.1-10% (w/v), orabout 10% (w/v).
 54. The method of claim 50, wherein the pharmaceuticalcomposition has a pH of from about 6 to about
 8. 55. The method of claim54, wherein the net charge of the self-assembling peptide in thepharmaceutical composition is greater than or equal to +1 or less thanor equal to −1.
 56. The method of claim 50, wherein the concentration ofthe self-assembling peptide in the pharmaceutical composition is fromabout 0.01% (w/v) to about 10% (w/v), about 0.1% (w/v) to about 5%(w/v), about 0.5% (w/v) to about 1.5% (w/v), or about 1% (w/v).
 57. Themethod of claim 50, wherein the pharmaceutical composition furthercomprises an isolated cell.
 58. The method of claim 57, wherein theisolated cell is a mammalian cell selected from the group consisting of:an immune cell, a stem cell, chondrocyte progenitor cells, pancreaticprogenitor cells, myoblasts, fibroblasts, keratinocytes, neuronal cells,glial cells, astrocytes, pre-adipocytes, adipocytes, vascularendothelial cells, endothelial progenitor cells, mesenchymal cells,neural stem cells, immune cells, (e.g., B-cells and T-cells), smoothmuscle progenitor cells, cardiac myocytes, fetal dermal fibroblasts,epidermal keratinocytes, myoblasts, and capillary endothelial cells. 59.The method of claim 50, wherein the pharmaceutical composition furthercomprises a bioactive agent.
 60. The method of claim 59, wherein thebioactive agent is selected from the group consisting of a hormone, agrowth factor, insulin, an enzyme, an siRNA, an shRNA, an antisense-RNA,an antibiotic, an antibody, and an anti-inflammatory agent.
 61. Themethod of claim 50, wherein the pharmaceutical composition is an aqueoussolution.
 62. The method of claim 50, wherein the pharmaceuticalcomposition is a hydrogel.
 63. The method of claim 62, wherein thehydrogel is characterized by having a storage modulus of at least about10 Pascal (Pa).
 64. The method of claim 50, comprising providing thepharmaceutical composition in an article of manufacture, wherein thearticle is a syringe, a vial, an auto-injector, tubing, or a catheter.65. (canceled)
 66. (canceled)
 67. (canceled)
 68. (canceled) 69.(canceled)